Compositions and methods for the therapy and diagnosis of breast cancer

ABSTRACT

Compositions and methods for the therapy and diagnosis of cancer, particularly breast cancer, are disclosed. Illustrative compositions comprise one or more breast tumor polypeptides, immunogenic portions thereof, polynucleotides that encode such polypeptides, antigen presenting cell that expresses such polypeptides, and T cells that are specific for cells expressing such polypeptides. The disclosed compositions are useful, for example, in the diagnosis, prevention and/or treatment of diseases, particularly breast cancer.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to therapy and diagnosis ofcancer, such as breast cancer. The invention is more specificallyrelated to polypeptides, comprising at least a portion of a breast tumorprotein, and to polynucleotides encoding such polypeptides. Suchpolypeptides and polynucleotides are useful in pharmaceuticalcompositions, e.g., vaccines, and other compositions for the diagnosisand treatment of breast cancer.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Breast cancer is a significant health problem for women in the UnitedStates and throughout the world. Although advances have been made indetection and treatment of the disease, breast cancer remains the secondleading cause of cancer-related deaths in women, affecting more than180,000 women in the United States each year. For women in NorthAmerica, the life-time odds of getting breast cancer are one in eight.

2. Description of the Related Art

No vaccine or other universally successful method for the prevention ortreatment of breast cancer is currently available. Management of thedisease currently relies on a combination of early diagnosis (throughroutine breast screening procedures) and aggressive treatment, which mayinclude one or more of a variety of treatments such as surgery,radiotherapy, chemotherapy and hormone therapy. The course of treatmentfor a particular breast cancer is often selected based on a variety ofprognostic parameters, including an analysis of specific tumor markers.See, e.g., Porter-Jordan and Lippman, Breast Cancer 8:73-100 (1994).However, the use of established markers often leads to a result that isdifficult to interpret, and the high mortality observed in breast cancerpatients indicates that improvements are needed in the treatment,diagnosis and prevention of the disease.

Accordingly, there is a need in the art for improved methods for thetreatment and diagnosis of breast cancer. The present invention fulfillsthese needs and further provides other related advantages.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides polynucleotidecompositions comprising a sequence selected from the group consistingof:

(a) sequences provided in SEQ ID NO:1-61, 63-175, 178, 180, 182-468,474, 476, 477, 479, 482, 484, 486, 489-492, 504-506, 510-513, 520-533,548-550, 564, 566-569, and 576;

(b) complements of the sequences provided in SEQ ID NO:1-61, 63-175,178, 180, 182-468, 474, 476, 477, 479, 482, 484, 486, 489-492, 504-506,510-513, 520-533, 548-550, 564, 566-569, and 576;

(c) sequences consisting of at least 20 contiguous residues of asequence provided in SEQ ID NO:1-61, 63-175, 178, 180, 182-468, 474,476, 477, 479, 482, 484, 486, 489-492, 504-506, 510-513, 520-533,548-550, 564, 566-569, and 576;

(d) sequences that hybridize to a sequence provided in SEQ ID NO: 1-61,63-175, 178, 180, 182-468, 474, 476, 477, 479, 482, 484, 486, 489-492,504-506, 510-513, 520-533, 548-550, 564, 566-569, and 576, undermoderately stringent conditions;

(e) sequences having at least 75% identity to a sequence of SEQ IDNO:1-61, 63-175, 178, 180, 182-468, 474, 476, 477, 479, 482, 484, 486,489-492, 504-506, 510-513, 520-533, 548-550, 564, 566-569, and 576;

(f) sequences having at least 90% identity to a sequence of SEQ IDNO:1-61, 63-175, 178, 180, 182-468, 474, 476, 477, 479, 482, 484, 486,489-492, 504-506, 510-513, 520-533, 548-550, 564, 566-569, and 576; and

(g) degenerate variants of a sequence provided in SEQ ID NO: 1-61,63-175, 178, 180, 182-468, 474, 476, 477, 479, 482, 484, 486, 489-492,504-506, 510-513, 520-533, 548-550, 564, 566-569, and 576.

In one preferred embodiment, the polynucleotide compositions of theinvention are expressed in at least about 20%, more preferably in atleast about 30%, and most preferably in at least about 50% of breasttumors samples tested, at a level that is at least about 2-fold,preferably at least about 5-fold, and most preferably at least about10-fold higher than that for normal tissues.

The present invention, in another aspect, provides polypeptidecompositions comprising an amino acid sequence that is encoded by apolynucleotide sequence described above.

The present invention further provides polypeptide compositionscomprising an amino acid sequence selected from the group consisting ofsequences recited in SEQ ID NO:62, 176, 179, 181, 469-473, 475, 478,483, 485, 487, 488, 493-503, 507-509, 514-519, 534-547, 551-553, 565,570-573, and 577-627.

In certain preferred embodiments, the polypeptides and/orpolynucleotides of the present invention are immunogenic, i.e., they arecapable of eliciting an immune response, particularly a humoral and/orcellular immune response, as further described herein.

The present invention further provides fragments, variants and/orderivatives of the disclosed polypeptide and/or polynucleotidesequences, wherein the fragments, variants and/or derivatives preferablyhave a level of immunogenic activity of at least about 50%, preferablyat least about 70% and more preferably at least about 90% of the levelof immunogenic activity of a polypeptide sequence set forth in SEQ IDNO: 62, 176, 179, 181, 469-473, 475, 478, 483, 485, 487, 488, 493-503,507-509, 514-519, 534-547, 551-553, 565, 570-573, and 577-627 or apolypeptide sequence encoded by a polynucleotide sequence set forth inSEQ ID NO: 1-61, 63-175, 178, 180, 182-468, 474, 476, 477, 479, 482,484, 486, 489-492, 504-506, 510-513, 520-533, 548-550, 564, 566-569, and576.

The present invention further provides polynucleotides that encode apolypeptide described above, expression vectors comprising suchpolynucleotides and host cells transformed or transfected with suchexpression vectors.

Within other aspects, the present invention provides pharmaceuticalcompositions comprising a polypeptide or polynucleotide as describedabove and a physiologically acceptable carrier.

Within a related aspect of the present invention, the pharmaceuticalcompositions, e.g., vaccine compositions, are provided for prophylacticor therapeutic applications. Such compositions generally comprise animmunogenic polypeptide or polynucleotide of the invention and animmunostimulant, such as an adjuvant.

The present invention further provides pharmaceutical compositions thatcomprise: (a) an antibody or antigen-binding fragment thereof thatspecifically binds to a polypeptide of the present invention, or afragment thereof; and (b) a physiologically acceptable carrier.

Within further aspects, the present invention provides pharmaceuticalcompositions comprising: (a) an antigen presenting cell that expresses apolypeptide as described above and (b) a pharmaceutically acceptablecarrier or excipient. Illustrative antigen presenting cells includedendritic cells, macrophages, monocytes, fibroblasts and B cells.

Within related aspects, pharmaceutical compositions are provided thatcomprise: (a) an antigen presenting cell that expresses a polypeptide asdescribed above and (b) an immunostimulant.

The present invention further provides, in other aspects, fusionproteins that comprise at least one polypeptide as described above, aswell as polynucleotides encoding such fusion proteins, typically in theform of pharmaceutical compositions, e.g., vaccine compositions,comprising a physiologically acceptable carrier and/or animmunostimulant. The fusions proteins may comprise multiple immunogenicpolypeptides or portions/variants thereof, as described herein, and mayfurther comprise one or more polypeptide segments for facilitating theexpression, purification and/or immunogenicity of the polypeptide(s).

The present invention further provides, in other aspects, fusionproteins that comprise at least one polypeptide as described above, aswell as polynucleotides encoding such fusion proteins. Exemplary fusionproteins according to the present invention comprise a first amino acidportion and a second amino acid portion wherein the first amino acidportion includes 9 or more contiguous amino acids from mammaglobin asdepicted by amino acids 1-93 of SEQ ID NO:493 (SEQ ID NO:503); whereinthe second amino acid portion includes 9 or more contiguous amino acidsfrom B726P as depicted by SEQ ID NO:475, SEQ ID NO:469, or SEQ IDNO:176; and wherein the first amino acid portion is connected to eitherthe amino terminal or carboxy-terminal end of the second amino acidportion.

Still further embodiments of the present invention provide fusionproteins wherein said first amino acid portion is selected from thegroup consisting of: IDELKECFLNQTDETLSNVE (SEQ ID NO:496; amino acids59-78 of SEQ ID NO:493); TFNAIDELKECFLNQ (SEQ ID NO:497; amino acids55-69 of SEQ ID NO:493); SQHCYAGSGCPLLENVISKTI (SEQ ID NO:498; aminoacids 13-33 of SEQ ID NO:493); EYKELLQEFIDDNATTNAID (SEQ ID NO:499;amino acids 41-60 of SEQ ID NO:493); KLLMVLMLA (SEQ ID NO:500; aminoacids 2-10 of SEQ ID NO:493); QEFIDDNATTNAI (SEQ ID NO:501; amino acids47-59 of SEQ ID NO:493); LKECFLNQTDETL (SEQ ID NO:502; amino acids 62-74of SEQ ID NO:493), and any one of the amino acid sequences set forth inSEQ ID NO:578-593.

Alternative embodiments provide fusion proteins wherein the second aminoacid portion includes 9 or more contiguous amino acids encoded by (1)the combined upstream and downstream open reading frame (ORF) of B726Pas depicted in SEQ ID NO:475; (2) the upstream ORF of B726P as depictedin SEQ ID NO:469; and (3) the downstream ORF of B726P as depicted in SEQID NO:176. Fusion proteins according to the present invention may alsocomprise a second amino acid portion that includes 9 or more contiguousamino acids from the amino acid sequence depicted by amino acids 1-129of SEQ ID NO:475. Still additional exemplary fusion proteins aredepicted herein by SEQ ID NO:493, SEQ ID NO:494, and SEQ ID NO:495.

Fusion proteins are provided wherein the mammaglobin amino acid portionis connected to the amino-terminus of the B726P amino acid portion whileother fusion proteins are provided wherein the mammaglobin amino acidportion is connected to the carboxy-terminus of the B726P amino acidportion. The connection between the mammaglobin amino acid portion andthe B726P portion may be a covalent bond. Additionally, a stretch ofamino acids either unrelated or related to either mammaglobin and/orB726P may be incorporated between or either amino- or carboxy-terminalto either the mammaglobin and/or B726P amino acid portion.

The present invention also provides isolated polynucleotides that encodeany of the fusion proteins that are specifically disclosed herein aswell as those fusion proteins that may be accomplished with routineexperimentation by the ordinarily skilled artisan.

Within further aspects, the present invention provides methods forstimulating an immune response in a patient, preferably a T cellresponse in a human patient, comprising administering a pharmaceuticalcomposition described herein. The patient may be afflicted with breastcancer, in which case the methods provide treatment for the disease, orpatient considered at risk for such a disease may be treatedprophylactically.

Within further aspects, the present invention provides methods forinhibiting the development of a cancer in a patient, comprisingadministering to a patient a pharmaceutical composition as recitedabove. The patient may be afflicted with breast cancer, in which casethe methods provide treatment for the disease, or patient considered atrisk for such a disease may be treated prophylactically.

The present invention further provides, within other aspects, methodsfor removing tumor cells from a biological sample, comprising contactinga biological sample with T cells that specifically react with apolypeptide of the present invention, wherein the step of contacting isperformed under conditions and for a time sufficient to permit theremoval of cells expressing the protein from the sample.

Within related aspects, methods are provided for inhibiting thedevelopment of a cancer in a patient, comprising administering to apatient a biological sample treated as described above.

Methods are further provided, within other aspects, for stimulatingand/or expanding T cells specific for a polypeptide of the presentinvention, comprising contacting T cells with one or more of: (i) apolypeptide as described above; (ii) a polynucleotide encoding such apolypeptide; and/or (iii) an antigen presenting cell that expresses sucha polypeptide; under conditions and for a time sufficient to permit thestimulation and/or expansion of T cells. Isolated T cell populationscomprising T cells prepared as described above are also provided.

Within further aspects, the present invention provides methods forinhibiting the development of a cancer in a patient, comprisingadministering to a patient an effective amount of a T cell population asdescribed above.

The present invention further provides methods for inhibiting thedevelopment of a cancer in a patient, comprising the steps of: (a)incubating CD4⁺ and/or CD8⁺ T cells isolated from a patient with one ormore of: (i) a polypeptide comprising at least an immunogenic portion ofpolypeptide disclosed herein; (ii) a polynucleotide encoding such apolypeptide; and (iii) an antigen-presenting cell that expressed such apolypeptide; and (b) administering to the patient an effective amount ofthe proliferated T cells, and thereby inhibiting the development of acancer in the patient. Proliferated cells may, but need not, be clonedprior to administration to the patient.

Within further aspects, the present invention provides methods fordetermining the presence or absence of a cancer, preferably a breastcancer, in a patient comprising: (a) contacting a biological sampleobtained from a patient with a binding agent that binds to a polypeptideas recited above; (b) detecting in the sample an amount of polypeptidethat binds to the binding agent; and (c) comparing the amount ofpolypeptide with a predetermined cut-off value, and therefromdetermining the presence or absence of a cancer in the patient. Withinpreferred embodiments, the binding agent is an antibody, more preferablya monoclonal antibody.

The present invention also provides, within other aspects, methods formonitoring the progression of a cancer in a patient. Such methodscomprise the steps of: (a) contacting a biological sample obtained froma patient at a first point in time with a binding agent that binds to apolypeptide as recited above; (b) detecting in the sample an amount ofpolypeptide that binds to the binding agent; (c) repeating steps (a) and(b) using a biological sample obtained from the patient at a subsequentpoint in time; and (d) comparing the amount of polypeptide detected instep (c) with the amount detected in step (b) and therefrom monitoringthe progression of the cancer in the patient.

The present invention further provides, within other aspects, methodsfor determining the presence or absence of a cancer in a patient,comprising the steps of: (a) contacting a biological sample obtainedfrom a patient with an oligonucleotide that hybridizes to apolynucleotide that encodes a polypeptide of the present invention; (b)detecting in the sample a level of a polynucleotide, preferably mRNA,that hybridizes to the oligonucleotide; and (c) comparing the level ofpolynucleotide that hybridizes to the oligonucleotide with apredetermined cut-off value, and therefrom determining the presence orabsence of a cancer in the patient. Within certain embodiments, theamount of mRNA is detected via polymerase chain reaction using, forexample, at least one oligonucleotide primer that hybridizes to apolynucleotide encoding a polypeptide as recited above, or a complementof such a polynucleotide. Within other embodiments, the amount of mRNAis detected using a hybridization technique, employing anoligonucleotide probe that hybridizes to a polynucleotide that encodes apolypeptide as recited above, or a complement of such a polynucleotide.

In related aspects, methods are provided for monitoring the progressionof a cancer in a patient, comprising the steps of: (a) contacting abiological sample obtained from a patient with an oligonucleotide thathybridizes to a polynucleotide that encodes a polypeptide of the presentinvention; (b) detecting in the sample an amount of a polynucleotidethat hybridizes to the oligonucleotide; (c) repeating steps (a) and (b)using a biological sample obtained from the patient at a subsequentpoint in time; and (d) comparing the amount of polynucleotide detectedin step (c) with the amount detected in step (b) and therefrommonitoring the progression of the cancer in the patient.

Within further aspects, the present invention provides antibodies, suchas monoclonal antibodies, that bind to a polypeptide as described above,as well as diagnostic kits comprising such antibodies. Diagnostic kitscomprising one or more oligonucleotide probes or primers as describedabove are also provided.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE IDENTIFIERS

FIG. 1 shows the results of a Northern blot of the clone SYN18C6 (SEQ IDNO:40).

FIG. 2 shows the results of an IFN-gamma ELISPOT assay demonstratingthat the B726P-specific CTL clone recognizes and lyses breast tumor celllines expressing B726P.

SEQ ID NO:1 is the determined cDNA sequence of JBT2.

SEQ ID NO:2 is the determined cDNA sequence of JBT6.

SEQ ID NO:3 is the determined cDNA sequence of JBT7.

SEQ ID NO:4 is the determined cDNA sequence of JBT10.

SEQ ID NO:5 is the determined cDNA sequence of JBT13.

SEQ ID NO:6 is the determined cDNA sequence of JBT14.

SEQ ID NO:7 is the determined cDNA sequence of JBT15.

SEQ ID NO:8 is the determined cDNA sequence of JBT16.

SEQ ID NO:9 is the determined cDNA sequence of JBT17.

SEQ ID NO:10 is the determined cDNA sequence of JBT22.

SEQ ID NO:11 is the determined cDNA sequence of JBT25.

SEQ ID NO:12 is the determined cDNA sequence of JBT28.

SEQ ID NO:13 is the determined cDNA sequence of JBT32.

SEQ ID NO:14 is the determined cDNA sequence of JBT33.

SEQ ID NO:15 is the determined cDNA sequence of JBT34.

SEQ ID NO:16 is the determined cDNA sequence of JBT36.

SEQ ID NO:17 is the determined cDNA sequence of JBT37.

SEQ ID NO:18 is the determined cDNA sequence of JBT51.

SEQ ID NO:19 is the determined cDNA sequence of JBTT1.

SEQ ID NO:20 is the determined cDNA sequence of JBTT7.

SEQ ID NO:21 is the determined cDNA sequence of JBTT11.

SEQ ID NO:22 is the determined cDNA sequence of JBTT14.

SEQ ID NO:23 is the determined cDNA sequence of JBTT18.

SEQ ID NO:24 is the determined cDNA sequence of JBTT19.

SEQ ID NO:25 is the determined cDNA sequence of JBTT20.

SEQ ID NO:26 is the determined cDNA sequence of JBTT21.

SEQ ID NO:27 is the determined cDNA sequence of JBTT22.

SEQ ID NO:28 is the determined cDNA sequence of JBTT28.

SEQ ID NO:29 is the determined cDNA sequence of JBTT29.

SEQ ID NO:30 is the determined cDNA sequence of JBTT33.

SEQ ID NO:31 is the determined cDNA sequence of JBTT37.

SEQ ID NO:32 is the determined cDNA sequence of JBTT38.

SEQ ID NO:33 is the determined cDNA sequence of JBTT47.

SEQ ID NO:34 is the determined cDNA sequence of JBTT48.

SEQ ID NO:35 is the determined cDNA sequence of JBTT50.

SEQ ID NO:36 is the determined cDNA sequence of JBTT51.

SEQ ID NO:37 is the determined cDNA sequence of JBTT52.

SEQ ID NO:38 is the determined cDNA sequence of JBTT54.

SEQ ID NO:39 is the determined cDNA sequence of SYN17F4.

SEQ ID NO:40 is the determined cDNA sequence of SYN18C6 (also known asB709P).

SEQ ID NO:41 is the determined cDNA sequence of SYN19A2.

SEQ ID NO:42 is the determined cDNA sequence of SYN19C8.

SEQ ID NO:43 is the determined cDNA sequence of SYN20A12.

SEQ ID NO:44 is the determined cDNA sequence of SYN20G6.

SEQ ID NO:45 is the determined cDNA sequence of SYN20G6-2.

SEQ ID NO:46 is the determined cDNA sequence of SYN21B9.

SEQ ID NO:47 is the determined cDNA sequence of SYN21B9-2.

SEQ ID NO:48 is the determined cDNA sequence of SYN21C10.

SEQ ID NO:49 is the determined cDNA sequence of SYN21G10.

SEQ ID NO:50 is the determined cDNA sequence of SYN21G10-2.

SEQ ID NO:51 is the determined cDNA sequence of SYN21G11.

SEQ ID NO:52 is the determined cDNA sequence of SYN21G11-2.

SEQ ID NO:53 is the determined cDNA sequence of SYN21H8.

SEQ ID NO:54 is the determined cDNA sequence of SYN22A10.

SEQ ID NO:55 is the determined cDNA sequence of SYN22A10-2.

SEQ ID NO:56 is the determined cDNA sequence of SYN22A12.

SEQ ID NO:57 is the determined cDNA sequence of SYN22A2 (also referredto as B718P).

SEQ ID NO:58 is the determined cDNA sequence of SYN22B4.

SEQ ID NO:59 is the determined cDNA sequence of SYN22C2.

SEQ ID NO:60 is the determined cDNA sequence of SYN22E10.

SEQ ID NO:61 is the determined cDNA sequence of SYN22F2.

SEQ ID NO:62 is a predicted amino acid sequence for SYN18C6 (also knownas B709P).

SEQ ID NO:63 is the determined cDNA sequence of B723P.

SEQ ID NO:64 is the determined cDNA sequence for B724P.

SEQ ID NO:65 is the determined cDNA sequence of B770P.

SEQ ID NO:66 is the determined cDNA sequence of B716P.

SEQ ID NO:67 is the determined cDNA sequence of B725P.

SEQ ID NO:68 is the determined cDNA sequence of B717P.

SEQ ID NO:69 is the determined cDNA sequence of B771P.

SEQ ID NO:70 is the determined cDNA sequence of B722P.

SEQ ID NO:71 is the determined cDNA sequence of B726P.

SEQ ID NO:72 is the determined cDNA sequence of B727P.

SEQ ID NO:73 is the determined cDNA sequence of B728P.

SEQ ID NO:74-87 are the determined cDNA sequences of isolated cloneswhich show homology to known sequences.

SEQ ID NO:88 is the determined cDNA sequence of 13053.

SEQ ID NO:89 is the determined cDNA sequence of 13057.

SEQ ID NO:90 is the determined cDNA sequence of 13059.

SEQ ID NO:91 is the determined cDNA sequence of 13065.

SEQ ID NO:92 is the determined cDNA sequence of 13067.

SEQ ID NO:93 is the determined cDNA sequence of 13068.

SEQ ID NO:94 is the determined cDNA sequence of 13071.

SEQ ID NO:95 is the determined cDNA sequence of 13072.

SEQ ID NO:96 is the determined cDNA sequence of 13073.

SEQ ID NO:97 is the determined cDNA sequence of 13075.

SEQ ID NO:98 is the determined cDNA sequence of 13078.

SEQ ID NO:99 is the determined cDNA sequence of 13079.

SEQ ID NO:100 is the determined cDNA sequence of 13081.

SEQ ID NO:101 is the determined cDNA sequence of 13082.

SEQ ID NO:102 is the determined cDNA sequence of 13092.

SEQ ID NO:103 is the determined cDNA sequence of 13097.

SEQ ID NO:104 is the determined cDNA sequence of 13101.

SEQ ID NO:105 is the determined cDNA sequence of 13102.

SEQ ID NO:106 is the determined cDNA sequence of 13119.

SEQ ID NO:107 is the determined cDNA sequence of 13131.

SEQ ID NO:108 is the determined cDNA sequence of 13133.

SEQ ID NO:109 is the determined cDNA sequence of 13135.

SEQ ID NO:110 is the determined cDNA sequence of 13139.

SEQ ID NO:111 is the determined cDNA sequence of 13140.

SEQ ID NO:112 is the determined cDNA sequence of 13146.

SEQ ID NO:113 is the determined cDNA sequence of 13147.

SEQ ID NO:114 is the determined cDNA sequence of 13148.

SEQ ID NO:115 is the determined cDNA sequence of 13149.

SEQ ID NO:116 is the determined cDNA sequence of 13151.

SEQ ID NO:117 is the determined cDNA sequence of 13051

SEQ ID NO:118 is the determined cDNA sequence of 13052

SEQ ID NO:119 is the determined cDNA sequence of 13055

SEQ ID NO:120 is the determined cDNA sequence of 13058

SEQ ID NO:121 is the determined cDNA sequence of 13062

SEQ ID NO:122 is the determined cDNA sequence of 13064

SEQ ID NO:123 is the determined cDNA sequence of 13080

SEQ ID NO:124 is the determined cDNA sequence of 13093

SEQ ID NO:125 is the determined cDNA sequence of 13094

SEQ ID NO:126 is the determined cDNA sequence of 13095

SEQ ID NO:127 is the determined cDNA sequence of 13096

SEQ ID NO:128 is the determined cDNA sequence of 13099

SEQ ID NO:129 is the determined cDNA sequence of 13100

SEQ ID NO:130 is the determined cDNA sequence of 13103

SEQ ID NO:131 is the determined cDNA sequence of 13106

SEQ ID NO:132 is the determined cDNA sequence of 13107

SEQ ID NO:133 is the determined cDNA sequence of 13108

SEQ ID NO:134 is the determined cDNA sequence of 13121

SEQ ID NO:135 is the determined cDNA sequence of 13126

SEQ ID NO:136 is the determined cDNA sequence of 13129

SEQ ID NO:137 is the determined cDNA sequence of 13130

SEQ ID NO:138 is the determined cDNA sequence of 13134

SEQ ID NO:139 is the determined cDNA sequence of 13141

SEQ ID NO:140 is the determined cDNA sequence of 13142

SEQ ID NO:141 is the determined cDNA sequence of 14376

SEQ ID NO:142 is the determined cDNA sequence of 14377

SEQ ID NO:143 is the determined cDNA sequence of 14383

SEQ ID NO:144 is the determined cDNA sequence of 14384

SEQ ID NO:145 is the determined cDNA sequence of 14387

SEQ ID NO:146 is the determined cDNA sequence of 14392

SEQ ID NO:147 is the determined cDNA sequence of 14394

SEQ ID NO:148 is the determined cDNA sequence of 14398

SEQ ID NO:149 is the determined cDNA sequence of 14401

SEQ ID NO:150 is the determined cDNA sequence of 14402

SEQ ID NO:151 is the determined cDNA sequence of 14405

SEQ ID NO:152 is the determined cDNA sequence of 14409

SEQ ID NO:153 is the determined cDNA sequence of 14412

SEQ ID NO:154 is the determined cDNA sequence of 14414

SEQ ID NO:155 is the determined cDNA sequence of 14415

SEQ ID NO:156 is the determined cDNA sequence of 14416

SEQ ID NO:157 is the determined cDNA sequence of 14419

SEQ ID NO:158 is the determined cDNA sequence of 14426

SEQ ID NO:159 is the determined cDNA sequence of 14427

SEQ ID NO:160 is the determined cDNA sequence of 14375

SEQ ID NO:161 is the determined cDNA sequence of 14378

SEQ ID NO:162 is the determined cDNA sequence of 14379

SEQ ID NO:163 is the determined cDNA sequence of 14380

SEQ ID NO:164 is the determined cDNA sequence of 14381

SEQ ID NO:165 is the determined cDNA sequence of 14382

SEQ ID NO:166 is the determined cDNA sequence of 14388

SEQ ID NO:167 is the determined cDNA sequence of 14399

SEQ ID NO:168 is the determined cDNA sequence of 14406

SEQ ID NO:169 is the determined cDNA sequence of 14407

SEQ ID NO:170 is the determined cDNA sequence of 14408

SEQ ID NO:171 is the determined cDNA sequence of 14417

SEQ ID NO:172 is the determined cDNA sequence of 14418

SEQ ID NO:173 is the determined cDNA sequence of 14423

SEQ ID NO:174 is the determined cDNA sequence of 14424

SEQ ID NO:175 is the determined cDNA sequence of B726P-20

SEQ ID NO:176 is the predicted amino acid sequence of B726P-20 (alsoreferred to as B726P downstream ORF)

SEQ ID NO:177 is a PCR primer

SEQ ID NO:178 is the determined cDNA sequence of B726P-74

SEQ ID NO:179 is the predicted amino acid sequence of B726P-74

SEQ ID NO:180 is the determined cDNA sequence of B726P-79

SEQ ID NO:181 is the predicted amino acid sequence of B726P-79

SEQ ID NO:182 is the determined cDNA sequence of 19439.1, showinghomology to the mammaglobin gene

SEQ ID NO:183 is the determined cDNA sequence of 19407.1, showinghomology to the human keratin gene

SEQ ID NO:184 is the determined cDNA sequence of 19428.1, showinghomology to human chromosome 17 clone

SEQ ID NO:185 is the determined cDNA sequence of B808P (19408), showingno significant homology to any known gene

SEQ ID NO:186 is the determined cDNA sequence of 19460.1, showing nosignificant homology to any known gene

SEQ ID NO:187 is the determined cDNA sequence of 19419.1, showinghomology to Ig kappa light chain

SEQ ID NO:188 is the determined cDNA sequence of 19411.1, showinghomology to human alpha-1 collagen

SEQ ID NO:189 is the determined cDNA sequence of 19420.1, showinghomology to mus musculus proteinase-3

SEQ ID NO:190 is the determined cDNA sequence of 19432.1, showinghomology to human high motility group box

SEQ ID NO:191 is the determined cDNA sequence of 19412.1, showinghomology to the human plasminogen activator gene

SEQ ID NO:192 is the determined cDNA sequence of 19415.1, showinghomology to mitogen activated protein kinase

SEQ ID NO:193 is the determined cDNA sequence of 19409.1, showinghomology to the chondroitin sulfate proteoglycan protein

SEQ ID NO:194 is the determined cDNA sequence of 19406.1, showing nosignificant homology to any known gene

SEQ ID NO:195 is the determined cDNA sequence of 19421.1, showinghomology to human fibronectin

SEQ ID NO:196 is the determined cDNA sequence of 19426.1, showinghomology to the retinoic acid receptor responder 3

SEQ ID NO:197 is the determined cDNA sequence of 19425.1, showinghomology to MyD88 mRNA

SEQ ID NO:198 is the determined cDNA sequence of 19424.1, showinghomology to peptide transporter (TAP-1) mRNA

SEQ ID NO:199 is the determined cDNA sequence of 19429.1, showing nosignificant homology to any known gene

SEQ ID NO:200 is the determined cDNA sequence of 19435.1, showinghomology to human polymorphic epithelial mucin

SEQ ID NO:201 is the determined cDNA sequence of B813P (19434.1),showing homology to human GATA-3 transcription factor

SEQ ID NO:202 is the determined cDNA sequence of 19461.1, showinghomology to the human AP-2 gene

SEQ ID NO:203 is the determined cDNA sequence of 19450.1, showinghomology to DNA binding regulatory factor

SEQ ID NO:204 is the determined cDNA sequence of 19451.1, showinghomology to Na/H exchange regulatory co-factor

SEQ ID NO:205 is the determined cDNA sequence of 19462.1, showing nosignificant homology to any known gene

SEQ ID NO:206 is the determined cDNA sequence of 19455.1, showinghomology to human mRNA for histone HAS.Z

SEQ ID NO:207 is the determined cDNA sequence of 19459.1, showinghomology to PAC clone 179N16

SEQ ID NO:208 is the determined cDNA sequence of 19464.1, showing nosignificant homology to any known gene

SEQ ID NO:209 is the determined cDNA sequence of 19414.1, showinghomology to lipophilin B

SEQ ID NO:210 is the determined cDNA sequence of 19413.1, showinghomology to chromosome 17 clone hRPK.209_J_(—)20

SEQ ID NO:211 is the determined cDNA sequence of 19416.1, showing nosignificant homology to any known gene

SEQ ID NO:212 is the determined cDNA sequence of 19437.1, showinghomology to human clone 24976 mRNA

SEQ ID NO:213 is the determined cDNA sequence of 19449.1, showinghomology to mouse DNA for PG-M core protein

SEQ ID NO:214 is the determined cDNA sequence of 19446.1, showing nosignificant homology to any known gene

SEQ ID NO:215 is the determined cDNA sequence of 19452.1, showing nosignificant homology to any known gene

SEQ ID NO:216 is the determined cDNA sequence of 19483.1, showing nosignificant homology to any known gene

SEQ ID NO:217 is the determined cDNA sequence of 19526.1, showinghomology to human lipophilin C

SEQ ID NO:218 is the determined cDNA sequence of 19484.1, showinghomology to the secreted cement gland protein XAG-2

SEQ ID NO:219 is the determined cDNA sequence of 19470.1, showing nosignificant homology to any known gene

SEQ ID NO:220 is the determined cDNA sequence of 19469.1, showinghomology to the human HLA-DM gene

SEQ ID NO:221 is the determined cDNA sequence of 19482.1, showinghomology to the human pS2 protein gene

SEQ ID NO:222 is the determined cDNA sequence of B805P (19468.1),showing no significant homology to any known gene

SEQ ID NO:223 is the determined cDNA sequence of 19467.1, showinghomology to human thrombospondin mRNA

SEQ ID NO:224 is the determined cDNA sequence of 19498.1, showinghomology to the CDC2 gene involved in cell cycle control

SEQ ID NO:225 is the determined cDNA sequence of 19506.1, showinghomology to human cDNA for TREB protein

SEQ ID NO:226 is the determined cDNA sequence of B806P (19505.1),showing no significant homology to any known gene

SEQ ID NO:227 is the determined cDNA sequence of 19486.1, showinghomology to type I epidermal keratin

SEQ ID NO:228 is the determined cDNA sequence of 19510.1, showinghomology to glucose transporter for glycoprotein

SEQ ID NO:229 is the determined cDNA sequence of 19512.1, showinghomology to the human lysyl hydroxylase gene

SEQ ID NO:230 is the determined cDNA sequence of 19511.1, showinghomology to human palimotoyl-protein thioesterase

SEQ ID NO:231 is the determined cDNA sequence of 19508.1, showinghomology to human alpha enolase

SEQ ID NO:232 is the determined cDNA sequence of B807P (19509.1),showing no significant homology to any known gene

SEQ ID NO:233 is the determined cDNA sequence of B809P (19520.1),showing homology to clone 102D24 on chromosome 11q13.31

SEQ ID NO:234 is the determined cDNA sequence of 19507.1, showinghomology toprosome beta-subunit

SEQ ID NO:235 is the determined cDNA sequence of 19525.1, showinghomology to human pro-urokinase precursor

SEQ ID NO:236 is the determined cDNA sequence of 19513.1, showing nosignificant homology to any known gene

SEQ ID NO:237 is the determined cDNA sequence of 19517.1, showinghomology to human PAC 128M19 clone

SEQ ID NO:238 is the determined cDNA sequence of 19564.1, showinghomology to human cytochrome P450-IIB

SEQ ID NO:239 is the determined cDNA sequence of 19553.1, showinghomology to human GABA-A receptor pi subunit

SEQ ID NO:240 is the determined cDNA sequence of B811P (19575.1),showing no significant homology to any known gene

SEQ ID NO:241 is the determined cDNA sequence of B810P (19560.1),showing no significant homology to any known gene

SEQ ID NO:242 is the determined cDNA sequence of 19588.1, showinghomology to aortic carboxypetidase-like protein

SEQ ID NO:243 is the determined cDNA sequence of 19551.1, showinghomology to human BCL-1 gene

SEQ ID NO:244 is the determined cDNA sequence of 19567.1, showinghomology to human proteasome-related mRNA

SEQ ID NO:245 is the determined cDNA sequence of B803P (19583.1),showing no significant homology to any known gene

SEQ ID NO:246 is the determined cDNA sequence of B812P (19587.1),showing no significant homology to any known gene

SEQ ID NO:247 is the determined cDNA sequence of B802P (19392.2),showing homology to human chromosome 17

SEQ ID NO:248 is the determined cDNA sequence of 19393.2, showinghomology to human nicein B2 chain

SEQ ID NO:249 is the determined cDNA sequence of 19398.2, human MHCclass II DQ alpha mRNA

SEQ ID NO:250 is the determined cDNA sequence of B804P (19399.2),showing homology to human Xp22 BAC GSHB-184P14

SEQ ID NO:251 is the determined cDNA sequence of 19401.2, showinghomology to human ikB kinase-b gene

SEQ ID NO:252 is the determined cDNA sequence of 20266, showing nosignificant homology to any known gene

SEQ ID NO:253 is the determined cDNA sequence of B826P (20270), showingno significant homology to any known gene

SEQ ID NO:254 is the determined cDNA sequence of 20274, showing nosignificant homology to any known gene

SEQ ID NO:255 is the determined cDNA sequence of 20276, showing nosignificant homology to any known gene

SEQ ID NO:256 is the determined cDNA sequence of 20277, showing nosignificant homology to any known gene

SEQ ID NO:257 is the determined cDNA sequence of B823P (20280), showingno significant homology to any known gene

SEQ ID NO:258 is the determined cDNA sequence of B821P (20281), showingno significant homology to any known gene

SEQ ID NO:259 is the determined cDNA sequence of B824P (20294), showingno significant homology to any known gene

SEQ ID NO:260 is the determined cDNA sequence of 20303, showing nosignificant homology to any known gene

SEQ ID NO:261 is the determined cDNA sequence of B820P (20310), showingno significant homology to any known gene

SEQ ID NO:262 is the determined cDNA sequence of B825P (20336), showingno significant homology to any known gene

SEQ ID NO:263 is the determined cDNA sequence of B827P (20341), showingno significant homology to any known gene

SEQ ID NO:264 is the determined cDNA sequence of 20941, showing nosignificant homology to any known gene

SEQ ID NO:265 is the determined cDNA sequence of 20954, showing nosignificant homology to any known gene

SEQ ID NO:266 is the determined cDNA sequence of 20961, showing nosignificant homology to any known gene

SEQ ID NO:267 is the determined cDNA sequence of 20965, showing nosignificant homology to any known gene

SEQ ID NO:268 is the determined cDNA sequence of 20975, showing nosignificant homology to any known gene

SEQ ID NO:269 is the determined cDNA sequence of 20261, showing homologyto Human p120 catenin

SEQ. ID NO:270 is the determined cDNA sequence of B822P (20262), showinghomology to Human membrane glycoprotein 4F2

SEQ ID NO:271 is the determined cDNA sequence of 20265, showing homologyto Human Na, K-ATPase Alpha 1

SEQ ID NO:272 is the determined cDNA sequence of 20267, showing homologyto Human heart HS 90, partial cds

SEQ ID NO:273 is the determined cDNA sequence of 20268, showing homologyto Human mRNA GPI-anchored protein p137

SEQ ID NO:274 is the determined cDNA sequence of 20271, showing homologyto Human cleavage stimulation factor 77 kDa subunit

SEQ ID NO:275 is the determined cDNA sequence of 20272, showing homologyto Human p190-B

SEQ ID NO:276 is the determined cDNA sequence of 20273, showing homologyto Human ribophorin

SEQ ID NO:277 is the determined cDNA sequence of 20278, showing homologyto Human ornithine amino transferase

SEQ ID NO:278 is the determined cDNA sequence of 20279, showing homologyto Human S-adenosylmethionine synthetase

SEQ ID NO:279 is the determined cDNA sequence of 20293, showing homologyto Human x inactivation transcript

SEQ ID NO:280 is the determined cDNA sequence of 20300, showing homologyto Human cytochrome p450

SEQ ID NO:281 is the determined cDNA sequence of 20305, showing homologyto Human elongation factor-1 alpha

SEQ ID NO:282 is the determined cDNA sequence of 20306, showing homologyto Human epithelial ets protein

SEQ ID NO:283 is the determined cDNA sequence of 20307, showing homologyto Human signal transducer mRNA

SEQ ID NO:284 is the determined cDNA sequence of 20313, showing homologyto Human GABA-A receptor pi subunit mRNA

SEQ ID NO:285 is the determined cDNA sequence of 20317, showing homologyto Human tyrosine phosphatase

SEQ ID NO:286 is the determined cDNA sequence of 20318, showing homologyto Human cathepsine B proteinase

SEQ ID NO:287 is the determined cDNA sequence of 20320, showing homologyto Human 2-phosphopyruvate-hydratase-alpha-enolase

SEQ ID NO:288 is the determined cDNA sequence of 20321, showing homologyto Human E-cadherin

SEQ ID NO:289 is the determined cDNA sequence of 20322, showing homologyto Human hsp86

SEQ ID NO:290 is the determined cDNA sequence of B828P (20326), showinghomology to Human x inactivation transcript

SEQ ID NO:291 is the determined cDNA sequence of 20333, showing homologyto Human chromatin regulator, SMARCA5

SEQ ID NO:292 is the determined cDNA sequence of 20335, showing homologyto Human sphingolipid activator protein 1

SEQ ID NO:293 is the determined cDNA sequence of 20337, showing homologyto Human hepatocyte growth factor activator inhibitor type 2

SEQ ID NO:294 is the determined cDNA sequence of 20338, showing homologyto Human cell adhesion molecule CD44

SEQ ID NO:295 is the determined cDNA sequence of 20340, showing homologyto Human nuclear factor (erythroid-derived)-like 1

SEQ ID NO:296 is the determined cDNA sequence of 20938, showing homologyto Human vinculin mRNA

SEQ ID NO:297 is the determined cDNA sequence of 20939, showing homologyto Human elongation factor EF-1-alpha

SEQ ID NO:298 is the determined cDNA sequence of 20940, showing homologyto Human nestin gene

SEQ ID NO:299 is the determined cDNA sequence of 20942, showing homologyto Human pancreatic ribonuclease

SEQ ID NO:300 is the determined cDNA sequence of 20943, showing homologyto Human transcobalamin I

SEQ ID NO:301 is the determined cDNA sequence of 20944, showing homologyto Human beta-tubulin

SEQ ID NO:302 is the determined cDNA sequence of 20946, showing homologyto Human HS1 protein

SEQ ID NO:303 is the determined cDNA sequence of 20947, showing homologyto Human cathepsin B

SEQ ID NO:304 is the determined cDNA sequence of 20948, showing homologyto Human testis enhanced gene transcript

SEQ ID NO:305 is the determined cDNA sequence of 20949, showing homologyto Human elongation factor EF-1-alpha

SEQ ID NO:306 is the determined cDNA sequence of 20950, showing homologyto Human ADP-ribosylation factor 3

SEQ ID NO:307 is the determined cDNA sequence of 20951, showing homologyto Human IFP53 or WRS for tryptophanyl-tRNA synthetase

SEQ ID NO:308 is the determined cDNA sequence of 20952, showing homologyto Human cyclin-dependent protein kinase

SEQ ID NO:309 is the determined cDNA sequence of 20957, showing homologyto Human alpha-tubulin isoform 1

SEQ ID NO:310 is the determined cDNA sequence of 20959, showing homologyto Human tyrosine phosphatase-61 bp deletion

SEQ ID NO:311 is the determined cDNA sequence of 20966, showing homologyto Human tyrosine phosphatase

SEQ ID NO:312 is the determined cDNA sequence of B830P (20976), showinghomology to Human nuclear factor NF 45

SEQ ID NO:313 is the determined cDNA sequence of B829P (20977), showinghomology to Human delta-6 fatty acid desaturase

SEQ ID NO:314 is the determined cDNA sequence of 20978, showing homologyto Human nuclear aconitase

SEQ ID NO:315 is the determined cDNA sequence of clone 23176.

SEQ ID NO:316 is the determined cDNA sequence of clone 23140.

SEQ ID NO:317 is the determined cDNA sequence of clone 23166.

SEQ ID NO:318 is the determined cDNA sequence of clone 23167.

SEQ ID NO:319 is the determined cDNA sequence of clone 23177.

SEQ ID NO:320 is the determined cDNA sequence of clone 23217.

SEQ ID NO:321 is the determined cDNA sequence of clone 23169.

SEQ ID NO:322 is the determined cDNA sequence of clone 23160.

SEQ ID NO:323 is the determined cDNA sequence of clone 23182.

SEQ ID NO:324 is the determined cDNA sequence of clone 23232.

SEQ ID NO:325 is the determined cDNA sequence of clone 23203.

SEQ ID NO:326 is the determined cDNA sequence of clone 23198.

SEQ ID NO:327 is the determined cDNA sequence of clone 23224.

SEQ ID NO:328 is the determined cDNA sequence of clone 23142.

SEQ ID NO:329 is the determined cDNA sequence of clone 23138.

SEQ ID NO:330 is the determined cDNA sequence of clone 23147.

SEQ ID NO:331 is the determined cDNA sequence of clone 23148.

SEQ ID NO:332 is the determined cDNA sequence of clone 23149.

SEQ ID NO:333 is the determined cDNA sequence of clone 23172.

SEQ ID NO:334 is the determined cDNA sequence of clone 23158.

SEQ ID NO:335 is the determined cDNA sequence of clone 23156.

SEQ ID NO:336 is the determined cDNA sequence of clone 23221.

SEQ ID NO:337 is the determined cDNA sequence of clone 23223.

SEQ ID NO:338 is the determined cDNA sequence of clone 23155.

SEQ ID NO:339 is the determined cDNA sequence of clone 23225.

SEQ ID NO:340 is the determined cDNA sequence of clone 23226.

SEQ ID NO:341 is the determined cDNA sequence of clone 23228.

SEQ ID NO:342 is the determined cDNA sequence of clone 23229.

SEQ ID NO:343 is the determined cDNA sequence of clone 23231.

SEQ ID NO:344 is the determined cDNA sequence of clone 23154.

SEQ ID NO:345 is the determined cDNA sequence of clone 23157.

SEQ ID NO:346 is the determined cDNA sequence of clone 23153.

SEQ ID NO:347 is the determined cDNA sequence of clone 23159.

SEQ ID NO:348 is the determined cDNA sequence of clone 23152.

SEQ ID NO:349 is the determined cDNA sequence of clone 23161.

SEQ ID NO:350 is the determined cDNA sequence of clone 23162.

SEQ ID NO:351 is the determined cDNA sequence of clone 23163.

SEQ ID NO:352 is the determined cDNA sequence of clone 23164.

SEQ ID NO:353 is the determined cDNA sequence of clone 23165.

SEQ ID NO:354 is the determined cDNA sequence of clone 23151.

SEQ ID NO:355 is the determined cDNA sequence of clone 23150.

SEQ ID NO:356 is the determined cDNA sequence of clone 23168.

SEQ ID NO:357 is the determined cDNA sequence of clone 23146.

SEQ ID NO:358 is the determined cDNA sequence of clone 23170.

SEQ ID NO:359 is the determined cDNA sequence of clone 23171.

SEQ ID NO:360 is the determined cDNA sequence of clone 23145.

SEQ ID NO:361 is the determined cDNA sequence of clone 23174.

SEQ ID NO:362 is the determined cDNA sequence of clone 23175.

SEQ ID NO:363 is the determined cDNA sequence of clone 23144.

SEQ ID NO:364 is the determined cDNA sequence of clone 23178.

SEQ ID NO:365 is the determined cDNA sequence of clone 23179.

SEQ ID NO:366 is the determined cDNA sequence of clone 23180.

SEQ ID NO:367 is the determined cDNA sequence of clone 23181.

SEQ ID NO:368 is the determined cDNA sequence of clone 23143

SEQ ID NO:369 is the determined cDNA sequence of clone 23183.

SEQ ID NO:370 is the determined cDNA sequence of clone 23184.

SEQ ID NO:371 is the determined cDNA sequence of clone 23185.

SEQ ID NO:372 is the determined cDNA sequence of clone 23186.

SEQ ID NO:373 is the determined cDNA sequence of clone 23187.

SEQ ID NO:374 is the determined cDNA sequence of clone 23190.

SEQ ID NO:375 is the determined cDNA sequence of clone 23189.

SEQ ID NO:376 is the determined cDNA sequence of clone 23202.

SEQ ID NO:378 is the determined cDNA sequence of clone 23191.

SEQ ID NO:379 is the determined cDNA sequence of clone 23188.

SEQ ID NO:380 is the determined cDNA sequence of clone 23194.

SEQ ID NO:381 is the determined cDNA sequence of clone 23196.

SEQ ID NO:382 is the determined cDNA sequence of clone 23195.

SEQ ID NO:383 is the determined cDNA sequence of clone 23193.

SEQ ID NO:384 is the determined cDNA sequence of clone 23199.

SEQ ID NO:385 is the determined cDNA sequence of clone 23200.

SEQ ID NO:386 is the determined cDNA sequence of clone 23192.

SEQ ID NO:387 is the determined cDNA sequence of clone 23201.

SEQ ID NO:388 is the determined cDNA sequence of clone 23141.

SEQ ID NO:389 is the determined cDNA sequence of clone 23139.

SEQ ID NO:390 is the determined cDNA sequence of clone 23204.

SEQ ID NO:391 is the determined cDNA sequence of clone 23205.

SEQ ID NO:392 is the determined cDNA sequence of clone 23206.

SEQ ID NO:393 is the determined cDNA sequence of clone 23207.

SEQ ID NO:394 is the determined cDNA sequence of clone 23208.

SEQ ID NO:395 is the determined cDNA sequence of clone 23209.

SEQ ID NO:396 is the determined cDNA sequence of clone 23210.

SEQ ID NO:397 is the determined cDNA sequence of clone 23211.

SEQ ID NO:398 is the determined cDNA sequence of clone 23212.

SEQ ID NO:399 is the determined cDNA sequence of clone 23214.

SEQ ID NO:400 is the determined cDNA sequence of clone 23215.

SEQ ID NO:401 is the determined cDNA sequence of clone 23216.

SEQ ID NO:402 is the determined cDNA sequence of clone 23137.

SEQ ID NO:403 is the determined cDNA sequence of clone 23218.

SEQ ID NO:404 is the determined cDNA sequence of clone 23220.

SEQ ID NO:405 is the determined cDNA sequence of clone 19462.

SEQ ID NO:406 is the determined cDNA sequence of clone 19430.

SEQ ID NO:407 is the determined cDNA sequence of clone 19407.

SEQ ID NO:408 is the determined cDNA sequence of clone 19448.

SEQ ID NO:409 is the determined cDNA sequence of clone 19447.

SEQ ID NO:410 is the determined cDNA sequence of clone 19426.

SEQ ID NO:411 is the determined cDNA sequence of clone 19441.

SEQ ID NO:412 is the determined cDNA sequence of clone 19454.

SEQ ID NO:413 is the determined cDNA sequence of clone 19463.

SEQ ID NO:414 is the determined cDNA sequence of clone 19419.

SEQ ID NO:415 is the determined cDNA sequence of clone 19434.

SEQ ID NO:416 is the determined extended cDNA sequence of B820P.

SEQ ID NO:417 is the determined extended cDNA sequence of B821P.

SEQ ID NO:418 is the determined extended cDNA sequence of B822P.

SEQ ID NO:419 is the determined extended cDNA sequence of B823P.

SEQ ID NO:420 is the determined extended cDNA sequence of B824P.

SEQ ID NO:421 is the determined extended cDNA sequence of B825P.

SEQ ID NO:422 is the determined extended cDNA sequence of B826P.

SEQ ID NO:423 is the determined extended cDNA sequence of B827P.

SEQ ID NO:424 is the determined extended cDNA sequence of B828P.

SEQ ID NO:425 is the determined extended cDNA sequence of B829P.

SEQ ID NO:426 is the determined extended cDNA sequence of B830P.

SEQ ID NO:427 is the determined cDNA sequence of clone 266B4.

SEQ ID NO:428 is the determined cDNA sequence of clone 22892.

SEQ ID NO:429 is the determined cDNA sequence of clone 266G3.

SEQ ID NO:430 is the determined cDNA sequence of clone 22890.

SEQ ID NO:431 is the determined cDNA sequence of clone 264B4.

SEQ ID NO:432 is the determined cDNA sequence of clone 22883.

SEQ ID NO:433 is the determined cDNA sequence of clone 22882.

SEQ ID NO:434 is the determined cDNA sequence of clone 22880.

SEQ ID NO:435 is the determined cDNA sequence of clone 263G1.

SEQ ID NO:436 is the determined cDNA sequence of clone 263G6.

SEQ ID NO:437 is the determined cDNA sequence of clone 262B2.

SEQ ID NO:438 is the determined cDNA sequence of clone 262B6.

SEQ ID NO:439 is the determined cDNA sequence of clone 22869.

SEQ ID NO:440 is the determined cDNA sequence of clone 21374.

SEQ ID NO:441 is the determined cDNA sequence of clone 21362.

SEQ ID NO:442 is the determined cDNA sequence of clone 21349.

SEQ ID NO:443 is the determined cDNA sequence of clone 21309.

SEQ ID NO:444 is the determined cDNA sequence of clone 21097.

SEQ ID NO:445 is the determined cDNA sequence of clone 21096.

SEQ ID NO:446 is the determined cDNA sequence of clone 21094.

SEQ ID NO:447 is the determined cDNA sequence of clone 21093.

SEQ ID NO:448 is the determined cDNA sequence of clone 21091.

SEQ ID NO:449 is the determined cDNA sequence of clone 21089.

SEQ ID NO:450 is the determined cDNA sequence of clone 21087.

SEQ ID NO:451 is the determined cDNA sequence of clone 21085.

SEQ ID NO:452 is the determined cDNA sequence of clone 21084.

SEQ ID NO:453 is a first partial cDNA sequence of clone 2BT1-40.

SEQ ID NO:454 is a second partial cDNA sequence of clone 2BT1-40.

SEQ ID NO:455 is the determined cDNA sequence of clone 21063.

SEQ ID NO:456 is the determined cDNA sequence of clone 21062.

SEQ ID NO:457 is the determined cDNA sequence of clone 21060.

SEQ ID NO:458 is the determined cDNA sequence of clone 21053.

SEQ ID NO:459 is the determined cDNA sequence of clone 21050.

SEQ ID NO:460 is the determined cDNA sequence of clone 21036.

SEQ ID NO:461 is the determined cDNA sequence of clone 21037.

SEQ ID NO:462 is the determined cDNA sequence of clone 21048.

SEQ ID NO:463 is a consensus DNA sequence of B726P (referred to asB726P-spliced_seq_B726P).

SEQ ID NO:464 is the determined cDNA sequence of a second splice form ofB726P (referred to as 27490.seq_B726P).

SEQ ID NO:465 is the determined cDNA sequence of a third splice form ofB726P (referred to as 27068.seq_B726P).

SEQ ID NO:466 is the determined cDNA sequence of a second splice form ofB726P (referred to as 23113.seq_B726P).

SEQ ID NO:467 is the determined cDNA sequence of a second splice form ofB726P (referred to as 23103.seq_B726P).

SEQ ID NO:468 is the determined cDNA sequence of a second splice form ofB726P (referred to as 19310.seq_B726P).

SEQ ID NO:469 is the predicted amino acid sequence encoded by theupstream ORF of SEQ ID NO:463.

SEQ ID NO:470 is the predicted amino acid sequence encoded by SEQ IDNO:464.

SEQ ID NO:471 is the predicted amino acid sequence encoded by SEQ IDNO:465.

SEQ ID NO:472 is the predicted amino acid sequence encoded by SEQ IDNO:466.

SEQ ID NO:473 is the predicted amino acid sequence encoded by SEQ IDNO:467.

SEQ ID NO:474 is the determined cDNA sequence for an alternative spliceform of B726P.

SEQ ID NO:475 is the amino acid sequence encoded by SEQ ID NO:474.

SEQ ID NO:476 is the isolated cDNA sequence of B720P.

SEQ ID NO:477 is the cDNA sequence of a known keratin gene.

SEQ ID NO:478 is the amino acid sequence encoded by SEQ ID NO:477.

SEQ ID NO:479 is the determined cDNA sequence for clone 19465.

SEQ ID NO:480 and 481 are PCR primers.

SEQ ID NO:482 is the cDNA sequence for the expressed downstream ORF ofB726P.

SEQ ID NO:483 is the amino acid sequence for the expressed recombinantdownstream ORF of B726P.

SEQ ID NO:484 is the determined full-length cDNA sequence for B720P.

SEQ ID NO:485 is the amino acid sequence encoded by SEQ ID NO:484.

SEQ ID NO:486 is the determined cDNA sequence of a truncated form ofB720P, referred to as B720P-tr.

SEQ ID NO:487 is the amino acid sequence of B720P-tr.

SEQ ID NO:488 is the amino acid sequence of a naturally processedepitope of B726P recognized by B726P-specific CTL.

SEQ ID NO:489 is a DNA sequence encoding the B726P epitope set forth inSEQ ID NO:488.

SEQ ID NO:490 is a DNA sequence encoding a fusion protein whereinmammaglobin is fused to the B726P combined upstream and downstream openreading frame (ORF) (the amino acid sequence of the B726P combined ORFis disclosed herein as SEQ ID NO:475 which is encoded by the DNAsequence of SEQ ID NO:474).

SEQ ID NO:491 is a DNA sequence encoding a fusion protein whereinmammaglobin is fused to the B726P upstream ORF (the amino acid sequenceof the B726P upstream ORF is disclosed herein as SEQ ID NO:469 which isencoded by the DNA sequence of SEQ ID NO:463).

SEQ ID NO:492 is a DNA sequence encoding a fusion protein whereinmammaglobin is fused to the B726P downstream ORF (the amino acidsequence of the B726P downstream ORF is disclosed herein as SEQ IDNO:176 which is encoded by the DNA sequence of SEQ ID NO:175).

SEQ ID NO:493 is the amino acid sequence encoded by the DNA sequence ofSEQ ID NO:490.

SEQ ID NO:494 is the amino acid sequence encoded by the DNA sequence ofSEQ ID NO:491.

SEQ ID NO:495 is the amino acid sequence encoded by the DNA sequence ofSEQ ID NO:492.

SEQ ID NO:496 is amino acids 59-78 of SEQ ID NO:493.

SEQ ID NO:497 is amino acids 55-69 of SEQ ID NO:493.

SEQ ID NO:498 is amino acids 13-33 of SEQ ID NO:493.

SEQ ID NO:499 is amino acids 41-60 of SEQ ID NO:493.

SEQ ID NO:500 is amino acids 2-10 of SEQ ID NO:493.

SEQ ID NO:501 is amino acids 47-59 of SEQ ID NO:493.

SEQ ID NO:502 is amino acids 62-74 of SEQ ID NO:493.

SEQ ID NO:503 is amino acids 1-93 of SEQ ID NO:493.

SEQ ID NO:504 is the full-length cDNA sequence for B718P.

SEQ ID NO:505 is the cDNA sequence of the open reading frame of B718Pincluding stop codon.

SEQ ID NO:506 is the cDNA sequence of the open reading frame of B718Pwithout stop codon.

SEQ ID NO:507 is the full-length amino acid sequence of B718P.

SEQ ID NO:508 represents amino acids 1-158 of SEQ ID NO:507.

SEQ ID NO:509 represents amino acids 159-243 of SEQ ID NO:509.

SEQ ID NO:510 is the entire cDNA sequence of the open reading frame,including stop codon, of a first variant of B723P, referred to asB723P-short.

SEQ ID NO:511 is the entire cDNA sequence of the open reading frame,without stop codon, of a first variant of B723P, referred to asB723P-short.

SEQ ID NO:512 is the entire cDNA sequence of the open reading frame,including stop codon, of a second variant of B723P, referred to asB723P-long.

SEQ ID NO:513 is the entire cDNA sequence of the open reading frame,without stop codon, of a second variant of B723P, referred to asB723P-long.

SEQ ID NO:514 is the amino acid sequence of B723P-short.

SEQ ID NO:515 is the amino acid sequence of B723P-long.

SEQ ID NO:516 is amino acids 1-197 of B723P-short.

SEQ ID NO:517 is amino acids 1-232 of B723P-long.

SEQ ID NO:518 is amino acids 198-243 of B723P-short.

SEQ ID NO:519 is amino acids 218-243 of B723P-short.

SEQ ID NO:520-533 are the DNA sequences of epitopes of B726P.

SEQ ID NO:534-547 are the amino acid sequences of epitopes of B726P.

SEQ ID NO:548 is the cDNA sequence of B726P Combined ORF coding_regionfor expression in E. coli.

SEQ ID NO:549 is the cDNA sequence of B726P Upstream ORF coding_regionfor expression in E. coli.

SEQ ID NO:550 is the cDNA sequence of B726P Downstream ORF coding regionfor expression in E. coli.

SEQ ID NO:551 is the amino acid sequence of B726P Downstream ORF encodedby the cDNA set forth in SEQ ID NO:550.

SEQ ID NO:552 is the amino acid sequence of B726P Upstream ORF with HIS,encoded by the cDNA set forth in SEQ ID NO:549.

SEQ ID NO:553 is the amino acid sequence of B726P Combined ORF correct,encoded by the cDNA set forth in SEQ ID NO:548.

SEQ ID NO:554-563 are PCR primers as described in Example 8.

SEQ ID NO:564 is the cDNA sequence for NY-BR-1, an extended sequence ofB726P.

SEQ ID NO:565 is the amino acid sequence for NY-BR-1, an extendedsequence of B726P, and encoded by the nucleotide sequence set forth inSEQ ID NO:564.

SEQ ID NO:566 is the cDNA sequence for B726P XC coding region withchanges.

SEQ ID NO:567 is the cDNA sequence for B726P XB clone 83686 with 2changes from the published NY-BR-1 sequence in SEQ ID NO:564.

SEQ ID NO:568 is the cDNA sequence for B726P XB clone 84330 with 4changes from the published NY-BR-1 sequence in SEQ ID NO:564.

SEQ ID NO:569 is the cDNA sequence for B726P XB clone 84328 with 3changes from the published NY-BR-1 sequence in SEQ ID NO:564.

SEQ ID NO:570 is the amino acid sequence for B726P XB clone 84328,encoded by the sequence set forth in SEQ ID NO:569.

SEQ ID NO:571 is the amino acid sequence for B726P XB clone 84330,encoded by the sequence set forth in SEQ ID NO:568.

SEQ ID NO:572 is the amino acid sequence for B726P XB clone 83686,encoded by the sequence set forth in SEQ ID NO:567.

SEQ ID NO:573 is the amino acid sequence for B726P XC, encoded by thesequence set forth in SEQ ID NO:566.

SEQ ID NO:574-575 are PCR primers as described in Example 12.

SEQ ID NO:576 is the full-length cDNA sequence for NY-BR-1.1.

SEQ ID NO:577 is the full-length amino acid sequence for NY-BR-1.1,encoded by the nucleotide sequence set forth in SEQ ID NO:576.

SEQ ID NO:578 is amino acids 289-308 of the B726P downstream ORF andcorresponds to the peptide recognized by the 220A2.1 antibody.

SEQ ID NO:579 is amino acids 225-244 of the B726P downstream ORF andcorresponds to the peptide recognized by the 220A19.1 antibody.

SEQ ID NO:580 is amino acids 232-252 of the B726P downstream ORF andcorresponds to the peptide recognized by the 220A19.1 and the 220A43antibodies.

SEQ ID NO:581 is amino acids 73-92 of the B726P downstream ORF andcorresponds to the peptide recognized by the 220A94.1 antibody.

SEQ ID NO:582 is amino acids 145-164 of the B726P downstream ORF andcorresponds to the peptide recognized by the 220A151.1 and 220A86antibodies.

SEQ ID NO:583 is amino acids 153-172 of the B726P downstream ORF andcorresponds to the peptide recognized by the 220A151.1 and 220A86antibodies.

SEQ ID NO:584 is amino acids 1-20 of the B726P downstream ORF andcorresponds to the peptide recognized by purified B726 polyclonalantibodies.

SEQ ID NO:585 is amino acids 9-28 of the B726P downstream ORF andcorresponds to the peptide recognized by purified B726 polyclonalantibodies.

SEQ ID NO:586 is amino acids 17-36 of the B726P downstream ORF andcorresponds to the peptide recognized by purified B726 polyclonalantibodies.

SEQ ID NO:587 is amino acids 24-44 of the B726P downstream ORF andcorresponds to the peptide recognized by purified B726 polyclonalantibodies.

SEQ ID NO:588 is amino acids 97-116 of the B726P downstream ORF andcorresponds to the peptide recognized by purified B726 polyclonalantibodies.

SEQ ID NO:589 is amino acids 105-124 of the B726P downstream ORF andcorresponds to the peptide recognized by purified B726 polyclonalantibodies.

SEQ ID NO:590 is amino acids 113-132 of the B726P downstream ORF andcorresponds to the peptide recognized by purified B726 polyclonalantibodies.

SEQ ID NO:591 is amino acids 121-140 of the B726P downstream ORF andcorresponds to the peptide recognized by purified B726 polyclonalantibodies.

SEQ ID NO:592 is amino acids 129-148 of the B726P downstream ORF andcorresponds to the peptide recognized by purified B726 polyclonalantibodies.

SEQ ID NO:593 is amino acids 137-156 of the B726P downstream ORF andcorresponds to the peptide recognized by purified B726 polyclonalantibodies.

SEQ ID NO:594 is the amino acid sequence of peptide #2732 andcorresponds to amino acids 1-20 of the B726P downstream ORF.

SEQ ID NO:595 is the amino acid sequence of peptide #2733 andcorresponds to amino acids 11-30 of the B726P downstream ORF.

SEQ ID NO:596 is the amino acid sequence of peptide #2734 andcorresponds to amino acids 21-40 of the B726P downstream ORF.

SEQ ID NO:597 is the amino acid sequence of peptide #2735 andcorresponds to amino acids 31-50 of the B726P downstream ORF.

SEQ ID NO:598 is the amino acid sequence of peptide #2736 andcorresponds to amino acids 41-60 of the B726P downstream ORF.

SEQ ID NO:599 is the amino acid sequence of peptide #2737 andcorresponds to amino acids 51-70 of the B726P downstream ORF.

SEQ ID NO:600 is the amino acid sequence of peptide #2738 andcorresponds to amino acids 61-80 of the B726P downstream ORF.

SEQ ID NO:601 is the amino acid sequence of peptide #2739 andcorresponds to amino acids 71-90 of the B726P downstream ORF.

SEQ ID NO:602 is the amino acid sequence of peptide #2740 andcorresponds to amino acids 81-100 of the B726P downstream ORF.

SEQ ID NO:603 is the amino acid sequence of peptide #2741 andcorresponds to amino acids 91-110 of the B726P downstream ORF.

SEQ ID NO:604 is the amino acid sequence of peptide #2742 andcorresponds to amino acids 101-120 of the B726P downstream ORF.

SEQ ID NO:605 is the amino acid sequence of peptide #2743 andcorresponds to amino acids 111-130 of the B726P downstream ORF.

SEQ ID NO:606 is the amino acid sequence of peptide #2744 andcorresponds to amino acids 121-140 of the B726P downstream ORF.

SEQ ID NO:607 is the amino acid sequence of peptide #2745 andcorresponds to amino acids 130-151 of the B726P downstream ORF.

SEQ ID NO:608 is the amino acid sequence of peptide #2746 andcorresponds to amino acids 141-160 of the B726P downstream ORF.

SEQ ID NO:609 is the amino acid sequence of peptide #2747 andcorresponds to amino acids 151-170 of the B726P downstream ORF.

SEQ ID NO:610 is the amino acid sequence of peptide #2748 andcorresponds to amino acids 161-180 of the B726P downstream ORF.

SEQ ID NO:611 is the amino acid sequence of peptide #2749 andcorresponds to amino acids 170-190 of the B726P downstream ORF.

SEQ ID NO:612 is the amino acid sequence of peptide #2750 andcorresponds to amino acids 181-200 of the B726P downstream ORF.

SEQ ID NO:613 is the amino acid sequence of peptide #2751 andcorresponds to amino acids 191-210 of the B726P downstream ORF.

SEQ ID NO:614 is the amino acid sequence of peptide #2752 andcorresponds to amino acids 201-220 of the B726P downstream ORF.

SEQ ID NO:615 is the amino acid sequence of peptide #2753 andcorresponds to amino acids 211-230 of the B726P downstream ORF.

SEQ ID NO:616 is the amino acid sequence of peptide #2765 andcorresponds to amino acids 221-240 of the B726P downstream ORF.

SEQ ID NO:617 is the amino acid sequence of peptide #2766 andcorresponds to amino acids 231-250 of the B726P downstream ORF.

SEQ ID NO:618 is the amino acid sequence of peptide #2767 andcorresponds to amino acids 240-260 of the B726P downstream ORF.

SEQ ID NO:619 is the amino acid sequence of peptide #2768 andcorresponds to amino acids 251-270 of the B726P downstream ORF.

SEQ ID NO:620 is the amino acid sequence of peptide #2769 andcorresponds to amino acids 261-280 of the B726P downstream ORF.

SEQ ID NO:621 is the amino acid sequence of peptide #2770 andcorresponds to amino acids 271-290 of the B726P downstream ORF.

SEQ ID NO:622 is the amino acid sequence of peptide #2771 andcorresponds to amino acids 281-300 of the B726P downstream ORF.

SEQ ID NO:623 is the amino acid sequence of peptide #2772 andcorresponds to amino acids 291-310 of the B726P downstream ORF.

SEQ ID NO:624 is the amino acid sequence of peptide #2773 andcorresponds to amino acids 298-317 of the B726P downstream ORF.

SEQ ID NO:625 is the amino acid sequence of peptide #3535 of B726P.

SEQ ID NO:626 is the amino acid sequence of peptide #3536 of B726P.

SEQ ID NO:627 is the amino acid sequence of peptide #3534 of B726P.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed generally to compositions and theiruse in the therapy and diagnosis of cancer, particularly breast cancer.As described further below, illustrative compositions of the presentinvention include, but are not restricted to, polypeptides, particularlyimmunogenic polypeptides, polynucleotides encoding such polypeptides,antibodies and other binding agents, antigen presenting cells (APCs) andimmune system cells (e.g., T cells).

The practice of the present invention will employ, unless indicatedspecifically to the contrary, conventional methods of virology,immunology, microbiology, molecular biology and recombinant DNAtechniques within the skill of the art, many of which are describedbelow for the purpose of illustration. Such techniques are explainedfully in the literature. See, e.g., Sambrook, et al. Molecular Cloning:A Laboratory Manual (2nd Edition, 1989); Maniatis et al. MolecularCloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach,vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed.,1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guideto Molecular Cloning (1984).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” include plural references unless the contentclearly dictates otherwise.

Polypeptide Compositions

As used herein, the term “polypeptide” “is used in its conventionalmeaning, i.e., as a sequence of amino acids. The polypeptides are notlimited to a specific length of the product; thus, peptides,oligopeptides, and proteins are included within the definition ofpolypeptide, and such terms may be used interchangeably herein unlessspecifically indicated otherwise. This term also does not refer to orexclude post-expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like, as well asother modifications known in the art, both naturally occurring andnon-naturally occurring. A polypeptide may be an entire protein, or asubsequence thereof. Particular polypeptides of interest in the contextof this invention are amino acid subsequences comprising epitopes, i.e.,antigenic determinants substantially responsible for the immunogenicproperties of a polypeptide and being capable of evoking an immuneresponse.

Particularly illustrative polypeptides of the present invention comprisethose encoded by a polynucleotide sequence set forth in any one of SEQID NO: 1-61, 63-175, 178, 180, 182-468, 474, 476, 477, 479, 482, 484,486, 489-492, 504-506, 510-513, 520-533, 548-550, 564, 566-569, and 576,or a sequence that hybridizes under moderately stringent conditions, or,alternatively, under highly stringent conditions, to a polynucleotidesequence set forth in any one of SEQ ID NO: 1-61, 63-175, 178, 180,182-468, 474, 476, 477, 479, 482, 484, 486, 489-492, 504-506, 510-513,520-533, 548-550, 564, 566-569, and 576. Certain other illustrativepolypeptides of the invention comprise amino acid sequences as set forthin any one of SEQ ID NO: 62, 176, 179, 181, 469-473, 475, 478, 483, 485,487, 488, 493-503, 507-509, 514-519, 534-547, 551-553, 565, 570-573, and577-627.

The polypeptides of the present invention are sometimes herein referredto as breast tumor proteins or breast tumor polypeptides, as anindication that their identification has been based at least in partupon their increased levels of expression in breast tumor samples. Thus,a “breast tumor polypeptide” or “breast tumor protein,” refers generallyto a polypeptide sequence of the present invention, or a polynucleotidesequence encoding such a polypeptide, that is expressed in a substantialproportion of breast tumor samples, for example preferably greater thanabout 20%, more preferably greater than about 30%, and most preferablygreater than about 50% or more of breast tumor samples tested, at alevel that is at least two fold, and preferably at least five fold,greater than the level of expression in normal tissues, as determinedusing a representative assay provided herein. A breast tumor polypeptidesequence of the invention, based upon its increased level of expressionin tumor cells, has particular utility both as a diagnostic marker aswell as a therapeutic target, as further described below.

In certain preferred embodiments, the polypeptides of the invention areimmunogenic, i.e., they react detectably within an immunoassay (such asan ELISA or T-cell stimulation assay) with antisera and/or T-cells froma patient with breast cancer. Screening for immunogenic activity can beperformed using techniques well known to the skilled artisan. Forexample, such screens can be performed using methods such as thosedescribed in Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988. In one illustrative example, apolypeptide may be immobilized on a solid support and contacted withpatient sera to allow binding of antibodies within the sera to theimmobilized polypeptide. Unbound sera may then be removed and boundantibodies detected using, for example, ¹²⁵I-labeled Protein A.

As would be recognized by the skilled artisan, immunogenic portions ofthe polypeptides disclosed herein are also encompassed by the presentinvention. An “immunogenic portion,” as used herein, is a fragment of animmunogenic polypeptide of the invention that itself is immunologicallyreactive (i.e., specifically binds) with the B-cells and/or T-cellsurface antigen receptors that recognize the polypeptide. Immunogenicportions may generally be identified using well known techniques, suchas those summarized in Paul, Fundamental Immunology, 3rd ed., 243-247(Raven Press, 1993) and references cited therein. Such techniquesinclude screening polypeptides for the ability to react withantigen-specific antibodies, antisera and/or T-cell lines or clones. Asused herein, antisera and antibodies are “antigen-specific” if theyspecifically bind to an antigen (i.e., they react with the protein in anELISA or other immunoassay, and do not react detectably with unrelatedproteins). Such antisera and antibodies may be prepared as describedherein, and using well-known techniques.

In one preferred embodiment, an immunogenic portion of a polypeptide ofthe present invention is a portion that reacts with antisera and/orT-cells at a level that is not substantially less than the reactivity ofthe full-length polypeptide (e.g., in an ELISA and/or T-cell reactivityassay). Preferably, the level of immunogenic activity of the immunogenicportion is at least about 50%, preferably at least about 70% and mostpreferably greater than about 90% of the immunogenicity for thefull-length polypeptide. In some instances, preferred immunogenicportions will be identified that have a level of immunogenic activitygreater than that of the corresponding full-length polypeptide, e.g.,having greater than about 100% or 150% or more immunogenic activity.

In certain other embodiments, illustrative immunogenic portions mayinclude peptides in, which an N-terminal leader sequence and/ortransmembrane domain have been deleted. Other illustrative immunogenicportions will contain a small N- and/or C-terminal deletion (e.g., 1-30amino acids, preferably 5-15 amino acids), relative to the matureprotein.

In another embodiment, a polypeptide composition of the invention mayalso comprise one or more polypeptides that are immunologically reactivewith T cells and/or antibodies generated against a polypeptide of theinvention, particularly a polypeptide having an amino acid sequencedisclosed herein, or to an immunogenic fragment or variant thereof.

In another embodiment of the invention, polypeptides are provided thatcomprise one or more polypeptides that are capable of eliciting T cellsand/or antibodies that are immunologically reactive with one or morepolypeptides described herein, or one or more polypeptides encoded bycontiguous nucleic acid sequences contained in the polynucleotidesequences disclosed herein, or immunogenic fragments or variantsthereof, or to one or more nucleic acid sequences which hybridize to oneor more of these sequences under conditions of moderate to highstringency.

The present invention, in another aspect, provides polypeptide fragmentscomprising at least about 5, 10, 15, 20, 25, 50, or 100 contiguous aminoacids, or more, including all intermediate lengths, of a polypeptidecompositions set forth herein, such as those set forth in SEQ ID NO: 62,176, 179,-181, 469-473, 475, 478, 483, 485, 487, 488, 493-503, 507-509,514-519, 534-547, 551-553, 565, 570-573, and 577-627, or those encodedby a polynucleotide sequence set forth in a sequence of SEQ ID NO: 1-61,63-175, 178, 180, 182-468, 474, 476, 477, 479, 482, 484, 486, 489-492,504-506, 510-513, 520-533, 548-550, 564, 566-569, and 576.

In another aspect, the present invention provides variants of thepolypeptide compositions described herein. Polypeptide variantsgenerally encompassed by the present invention will typically exhibit atleast about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more identity (determined as described below), along itslength, to a polypeptide sequences set forth herein.

In one preferred embodiment, the polypeptide fragments and variantsprovide by the present invention are immunologically reactive with anantibody and/or T-cell that reacts with a full-length polypeptidespecifically set for the herein.

In another preferred embodiment, the polypeptide fragments and variantsprovided by the present invention exhibit a level of immunogenicactivity of at least about 50%, preferably at least about 70%, and mostpreferably at least about 90% or more of that exhibited by a full-lengthpolypeptide sequence specifically set forth herein.

A polypeptide “variant,” as the term is used herein, is a polypeptidethat typically differs from a polypeptide specifically disclosed hereinin one or more substitutions, deletions, additions and/or insertions.Such variants may be naturally occurring or may be syntheticallygenerated, for example, by modifying one or more of the abovepolypeptide sequences of the invention and evaluating their immunogenicactivity as described herein and/or using any of a number of techniqueswell known in the art.

For example, certain illustrative variants of the polypeptides of theinvention include those in which one or more portions, such as anN-terminal leader sequence or transmembrane domain, have been removed.Other illustrative variants include variants in which a small portion(e.g., 1-30 amino acids, preferably 5-15 amino acids) has been removedfrom the N- and/or C-terminal of the mature protein.

In many instances, a variant will contain conservative substitutions. A“conservative substitution” is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. As described above, modifications may be made in thestructure of the polynucleotides and polypeptides of the presentinvention and still obtain a functional molecule that encodes a variantor derivative polypeptide with desirable characteristics, e.g., withimmunogenic characteristics. When it is desired to alter the amino acidsequence of a polypeptide to create an equivalent, or even an improved,immunogenic variant or portion of a polypeptide of the invention, oneskilled in the art will typically change one or more of the codons ofthe encoding DNA sequence according to Table 1.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated that various changes may bemade in the peptide sequences of the disclosed compositions, orcorresponding DNA sequences which encode said peptides withoutappreciable loss of their biological utility or activity. TABLE 1 AminoAcids Codons Alanine Ala A GGA GGG GGG GCU Cysteine Gys C UGC UGUAspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAG PhenylalaninePhe F UUG UUU Glycine Gly G GGA GGG GGG GGU Histidine His H GAG CAUIsoleucine lie I AUA AUG AUU Lysine Lys K AAA AAG Leucine Leu L UUA UUGCUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAG AAU ProlinePro P CCA CCC CCG CCU Glutamine Gin Q CAA CAG Arginine Arg R AGA AGG CGACGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UGU Threonine Thr T ACA ACCACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr YUAC UAU

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporated herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. Each amino acid has been assigned a hydropathicindex on the basis of its hydrophobicity and charge characteristics(Kyte and Doolittle, 1982). These values are: isoleucine (+4.5); valine(+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those within ±1 are particularly preferred, and thosewithin ±0.5 are even more particularly preferred. It is also understoodin the art that the substitution of like amino acids can be madeeffectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101(specifically incorporated herein by reference in its entirety), statesthat the greatest local average hydrophilicity of a protein, as governedby the hydrophilicity of its adjacent amino acids, correlates with abiological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). It isunderstood that an amino acid can be substituted for another having asimilar hydrophilicity value and still obtain a biologically equivalent,and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values arewithin ±2 is preferred, those within ±1 are particularly preferred, andthose within ±0;5 are even more particularly preferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions that take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

In addition, any polynucleotide may be further modified to increasestability in vivo. Possible modifications include, but are not limitedto, the addition of flanking sequences at the 5′ and/or 3′ ends; the useof phosphorothioate or 2′ O-methyl rather than phosphodiesteraselinkages in the backbone; and/or the inclusion of nontraditional basessuch as inosine, queosine and wybutosine, as well as acetyl- methyl-,thio- and other modified forms of adenine, cytidine, guanine, thymineand uridine.

Amino acid substitutions may further be made on the basis of similarityin polarity, charge, solubility, hydrophobicity, hydrophilicity and/orthe amphipathic nature of the residues. For example, negatively chargedamino acids include aspartic acid and glutamic acid; positively chargedamino acids include lysine and arginine; and amino acids with unchargedpolar head groups having similar hydrophilicity values include leucine,isoleucine and valine; glycine and alanine; asparagine and glutamine;and serine, threonine, phenylalanine and tyrosine. Other groups of aminoacids that may represent conservative changes include: (1) ala, pro,gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile,leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Avariant may also, or alternatively, contain nonconservative changes. Ina preferred embodiment, variant polypeptides differ from a nativesequence by substitution, deletion or addition of five amino acids orfewer. Variants may also (or alternatively) be modified by, for example,the deletion or addition of amino acids that have minimal influence onthe immunogenicity, secondary structure and hydropathic nature of thepolypeptide.

As noted above, polypeptides may comprise a signal (or leader) sequenceat the N-terminal end of the protein, which co-translationally orpost-translationally directs transfer of the protein. The polypeptidemay also be conjugated to a linker or other sequence for ease ofsynthesis, purification or identification of the polypeptide (e.g.,poly-His), or to enhance binding of the polypeptide to a solid support.For example, a polypeptide may be conjugated to an immunoglobulin Fcregion.

When comparing polypeptide sequences, two sequences are said to be“identical” if the sequence of amino acids in the two sequences is thesame when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA80:726-730.

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman (1981)Add. APL. Math 2:482, by the identity alignment algorithm of Needlemanand Wunsch (1970) J. Mol. Biol. 48:443, by the search for similaritymethods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT,BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or byinspection.

One preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al. (1977)Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol.215:403-410, respectively. BLAST and BLAST 2.0 can be used, for examplewith the parameters described herein, to determine percent sequenceidentity for the polynucleotides and polypeptides of the invention.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. For amino acid sequences,a scoring matrix can be used to calculate the cumulative score.Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment.

In one preferred approach, the “percentage of sequence identity” isdetermined by comparing two optimally aligned sequences over a window ofcomparison of at least 20 positions, wherein the portion of thepolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the referencesequence (i.e., the window size) and multiplying the results by 100 toyield the percentage of sequence identity.

Within other illustrative embodiments, a polypeptide may be a fusionpolypeptide that comprises multiple polypeptides as described herein, orthat comprises at least one polypeptide as described herein and anunrelated sequence, such as a known tumor protein. A fusion partner may,for example, assist in providing T helper epitopes (an immunologicalfusion partner), preferably T helper epitopes recognized by humans, ormay assist in expressing the protein (an expression enhancer) at higheryields than the native recombinant protein. Certain preferred fusionpartners are both immunological and expression enhancing fusionpartners. Other fusion partners may be selected so as to increase thesolubility of the polypeptide or to enable the polypeptide to betargeted to desired intracellular compartments. Still further fusionpartners include affinity tags, which facilitate purification of thepolypeptide.

Fusion polypeptides may generally be prepared using standard techniques,including chemical conjugation. Preferably, a fusion polypeptide isexpressed as a recombinant polypeptide, allowing the production ofincreased levels, relative to a non-fused polypeptide, in an expressionsystem. Briefly, DNA sequences encoding the polypeptide components maybe assembled separately, and ligated into an appropriate expressionvector. The 3′ end of the DNA sequence encoding one polypeptidecomponent is ligated, with or without a peptide linker, to the 5′ end ofa DNA sequence encoding the second polypeptide component so that thereading frames of the sequences are in phase. This permits translationinto a single fusion polypeptide that retains the biological activity ofboth component polypeptides.

A peptide linker sequence may be employed to separate the first andsecond polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and tertiary structures. Sucha peptide linker sequence is incorporated into the fusion polypeptideusing standard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might-react with the polypeptidefunctional epitopes. Preferred peptide linker sequences contain Gly, Asnand Ser residues. Other near neutral amino acids, such as Thr and Alamay also be used in the linker sequence. Amino acid sequences which maybe usefully employed as linkers include those disclosed in Maratea etal., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA83:8258-8262, 1986; U.S. Pat. No. 4,935,233 and U.S. Pat. No.4,751,180.The linker sequence may generally be from 1 to about 50 amino acids inlength. Linker sequences are not required when the first and secondpolypeptides have non-essential N-terminal amino acid regions that canbe used to separate the functional domains and prevent stericinterference.

The ligated DNA sequences are operably linked to suitabletranscriptional or translational regulatory elements. The regulatoryelements responsible for expression of DNA are located only 5′ to theDNA sequence encoding the first polypeptides. Similarly, stop codonsrequired to end translation and transcription termination signals areonly present 3′ to the DNA sequence encoding the second polypeptide.

The fusion polypeptide can comprise a polypeptide as described hereintogether with an unrelated immunogenic protein, such as an immunogenicprotein capable of eliciting a recall response. Examples of suchproteins include tetanus, tuberculosis and hepatitis proteins (see, forexample, Stoute et al. New Engl. J. Med., 336:86-91, 1997).

In one preferred embodiment, the immunological fusion partner is derivedfrom a Mycobacterium sp., such as a Mycobacterium tuberculosis-derivedRa12 fragment. Ra12 compositions and methods for their use in enhancingthe expression and/or immunogenicity of heterologouspolynucleotide/polypeptide sequences is described in U.S. PatentApplication 60/158,585, the disclosure of which is incorporated hereinby reference in its entirety. Briefly, Ra12 refers to a polynucleotideregion that is a subsequence of a Mycobacterium tuberculosis MTB32Anucleic acid. MTB32A is a serine protease of 32 KD molecular weightencoded by a gene in virulent and avirulent strains of M. tuberculosis.The nucleotide sequence and amino acid sequence of MTB32A have beendescribed (for example, U.S. Patent Application 60/158,585; see also,Skeiky et al., Infection and Immun. (1999) 67:3998-4007, incorporatedherein by reference). C-terminal fragments of the MTB32A coding sequenceexpress at high levels and remain as a soluble polypeptides throughoutthe purification process. Moreover, Ra12 may enhance the immunogenicityof heterologous immunogenic polypeptides with which it is fused. Onepreferred Ra12 fusion polypeptide comprises a 14 KD C-terminal fragmentcorresponding to amino acid residues 192 to 323 of MTB32A. Otherpreferred Ra12 polynucleotides generally comprise at least about 15consecutive nucleotides, at least about 30 nucleotides, at least about60 nucleotides, at least about 100 nucleotides, at least about 200nucleotides, or at least about 300 nucleotides that encode a portion ofa Ra12 polypeptide. Ra12 polynucleotides may comprise a native sequence(i.e., an endogenous sequence that encodes a Ra12 polypeptide or aportion thereof) or may comprise a variant of such a sequence. Ra12polynucleotide variants may contain one or more substitutions,additions, deletions and/or insertions such that the biological activityof the encoded fusion polypeptide is not substantially diminished,relative to a fusion polypeptide comprising a native Ra12 polypeptide.Variants preferably exhibit at least about 70% identity, more preferablyat least about 80% identity and most preferably at least about 90%identity to a polynucleotide sequence that encodes a native Ra12polypeptide or a portion thereof.

Within other preferred embodiments, an immunological fusion partner isderived from protein D, a surface protein of the gram-negative bacteriumHaemophilus influenza B (WO 91/18926). Preferably, a protein Dderivative comprises approximately the first third of the protein (e.g.,the first N-terminal 100-110 amino acids), and a protein D derivativemay be lipidated. Within certain preferred embodiments, the first 109residues of a Lipoprotein D fusion partner is included on the N-terminusto provide the polypeptide with additional exogenous T-cell epitopes andto increase the expression level in E. coli (thus functioning as anexpression enhancer). The lipid tail ensures optimal presentation of theantigen to antigen presenting cells. Other fusion partners include thenon-structural protein from influenzae virus, NS1 (hemaglutinin).Typically, the N-terminal 81 amino acids are used, although differentfragments that include T-helper epitopes may be used.

In another embodiment, the immunological fusion partner is the proteinknown as LYTA, or a portion thereof (preferably a C-terminal portion).LYTA is derived from Streptococcus pneumoniae, which synthesizes anN-acetyl-L-alanine amidase known as amidase LYTA (encoded by the LytAgene; Gene 43:265-292, 1986). LYTA is an autolysin that specificallydegrades certain bonds in the peptidoglycan backbone. The C-terminaldomain of the LYTA protein is responsible for the affinity to thecholine or to some choline analogues such as DEAE. This property hasbeen exploited for the development of E. coli C-LYTA expressing plasmidsuseful for expression of fusion proteins. Purification of hybridproteins containing the C-LYTA fragment at the amino terminus has beendescribed (see Biotechnology 10:795-798, 1992). Within a preferredembodiment, a repeat portion of LYTA may be incorporated into a fusionpolypeptide. A repeat portion is found in the C-terminal, regionstarting at residue 178. A particularly preferred repeat portionincorporates residues 188-305.

Yet another illustrative embodiment involves fusion polypeptides, andthe polynucleotides encoding them, wherein the fusion partner comprisesa targeting signal capable of directing a polypeptide to theendosomal/lysosomal compartment, as described in U.S. Pat. No.5,633,234. An immunogenic polypeptide of the invention, when fused withthis targeting signal, will associate more efficiently with MHC class IImolecules and thereby provide enhanced in vivo stimulation of CD4⁺T-cells specific for the polypeptide.

Polypeptides of the invention are prepared using any of a variety ofwell known synthetic and/or recombinant techniques, the latter of whichare further described below. Polypeptides, portions and other variantsgenerally less than about 150 amino acids can be generated by syntheticmeans, using techniques well known to those of ordinary skill in theart. In one illustrative example, such polypeptides are synthesizedusing any of the commercially available solid-phase techniques, such asthe Merrifield solid-phase synthesis method, where amino acids aresequentially added to a growing amino acid chain. See Merrifield, J. Am.Chem. Soc. 85:2149-2146, 1963. Equipment for automated synthesis ofpolypeptides is commercially available from suppliers such as PerkinElmer/Applied BioSystems Division (Foster City, Calif.), and may beoperated according to the manufacturer's instructions.

In general, polypeptide compositions (including fusion polypeptides) ofthe invention are isolated. An “isolated” polypeptide is one that isremoved from its original environment. For example, anaturally-occurring protein or polypeptide is isolated if it isseparated from some or all of the coexisting materials in the naturalsystem. Preferably, such polypeptides are also purified, e.g., are atleast about 90% pure, more preferably at least about 95% pure and mostpreferably at least about 99% pure.

Polynucleotide Compositions

The present invention, in other aspects, provides polynucleotidecompositions. The terms “DNA” and “polynucleotide” are used essentiallyinterchangeably herein to refer to a DNA molecule that has been isolatedfree of total genomic DNA of a particular species. “Isolated,” as usedherein, means that a polynucleotide is substantially away from othercoding sequences, and that the DNA molecule does not contain largeportions of unrelated coding DNA, such as large chromosomal fragments orother functional genes or polypeptide coding regions. Of course, thisrefers to the DNA molecule as originally isolated, and does not excludegenes or coding regions later added to the segment by the hand of man.

As will be understood by those skilled in the art, the polynucleotidecompositions of this invention can include genomic sequences,extra-genomic and plasmid-encoded sequences and smaller engineered genesegments that express, or may be adapted to express, proteins,polypeptides, peptides and the like. Such segments may be naturallyisolated, or modified synthetically by the hand of man.

As will be also recognized by the skilled artisan, polynucleotides ofthe invention may be single-stranded (coding or antisense) ordouble-stranded, and may be DNA (genomic, cDNA or synthetic) or RNAmolecules. RNA molecules may include HnRNA molecules, which containintrons and correspond to a DNA molecule in a one-to-one manner, andmRNA molecules, which do not contain introns. Additional coding ornon-coding sequences may, but need not, be present within apolynucleotide of the present invention, and a polynucleotide may, butneed not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence (i.e., an endogenoussequence that encodes a polypeptide/protein of the invention or aportion thereof) or may comprise a sequence that encodes a variant orderivative, preferably and immunogenic variant or derivative, of such asequence.

Therefore, according to another aspect of the present invention,polynucleotide compositions are provided that comprise some or all of apolynucleotide sequence set forth in any one of SEQ ID NO: 1-61, 63-175,178, 180, 182-468, 474, 476, 477, 479, 482, 484, 486, 489-492, 504-506,510-513, 520-533, 548-550, 564, 566-569, and 576, complements of apolynucleotide sequence set forth in any one of SEQ ID NO: 1-61, 63-175,178, 180, 182-468, 474, 476, 477, 479, 482, 484, 486, 489-492, 504-506,510-513, 520-533, 548-550, 564, 566-569, and 576, and degeneratevariants of a polynucleotide sequence set forth in any one of SEQ ID NO:1-61, 63-175, 178, 180, 182-468, 474, 476, 477, 479, 482, 484, 486,489-492, 504-506, 510-513, 520-533, 548-550, 564, 566-569, and 576. Incertain preferred embodiments, the polynucleotide sequences set forthherein encode immunogenic polypeptides, as described above.

In other related embodiments, the present invention providespolynucleotide variants having substantial identity to the sequencesdisclosed herein in SEQ ID NO: 1-61, 63-175, 178, 180, 182-468, 474,476, 477, 479, 482, 484, 486, 489-492,-504-506, 510-513, 520-533,548-550, 564, 566-569, and 576, for example those comprising at least70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% or higher, sequence identity compared to apolynuclebtide sequence of this invention using the methods describedherein, (e.g., BLAST analysis using standard parameters, as describedbelow). One skilled in this art will recognize that these values can beappropriately adjusted to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning and thelike.

Typically, polynucleotide variants will contain one or moresubstitutions, additions, deletions and/or insertions, preferably suchthat the immunogenicity of the polypeptide encoded by the variantpolynucleotide is not substantially diminished relative to a polypeptideencoded by a polynucleotide sequence specifically set forth herein). Theterm “variants” should also be understood to encompasses homologousgenes of xenogenic origin.

In additional embodiments, the present invention provides polynucleotidefragments comprising various lengths of contiguous stretches of sequenceidentical to or complementary to one or more of the sequences disclosedherein. For example, polynucleotides are provided by this invention thatcomprise at least about 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300,400, 500 or 1000 or more contiguous nucleotides of one or more of thesequences disclosed herein as well as all intermediate lengths therebetween. It will be readily understood that “intermediate lengths”, inthis context, means any length between the quoted values, such as 16,17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53,etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.; including allintegers through 200-500; 500-1,000, and the like.

In another embodiment of the invention, polynucleotide compositions areprovided that are capable of hybridizing under moderate to highstringency conditions to a polynucleotide sequence provided herein, or afragment thereof, or a complementary sequence thereof. Hybridizationtechniques are well known in the art of molecular biology. For purposesof illustration, suitable moderately stringent conditions for testingthe hybridization of a polynucleotide of this invention with otherpolynucleotides include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0mM EDTA (pH 8.0); hybridizing at 50° C.-60° C., 5×SSC, overnight;followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5×and 0.2×SSC containing 0.1% SDS. One skilled in the art will understandthat the stringency of hybridization can be readily manipulated, such asby altering the salt content of the hybridization solution and/or thetemperature at which the hybridization is performed. For example, inanother embodiment, suitable highly stringent hybridization conditionsinclude those described above, with the exception that the temperatureof hybridization is increased, e.g., to 60-65° C. or 65-70° C.

In certain preferred embodiments,-the polynucleotides described above,e.g., polynucleotide variants, fragments and hybridizing sequences,encode polypeptides that are immunologically cross-reactive with apolypeptide sequence specifically set forth herein. In other preferredembodiments, such polynucleotides encode polypeptides that have a levelof immunogenic activity of at least about 50%, preferably at least about70%, and more preferably at least about 90% of that for a polypeptidesequence specifically set forth herein.

The polynucleotides of the present invention, or fragments thereof,regardless of the length of the coding sequence itself, may be combinedwith other DNA sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length may be employed, with the total length preferablybeing limited by the ease of preparation and use in the intendedrecombinant DNA protocol. For example, illustrative polynucleotidesegments with total lengths of about 10,000, about 5000, about 3000,about 2,000, about 1,000, about 500, about 200, about 100, about 50 basepairs in length, and the like, (including all intermediate lengths) arecontemplated to be useful in many implementations of this invention.

When comparing polynucleotide sequences, two sequences are said to be“identical” if the sequence of nucleotides in the two sequences is thesame when aligned for maximum correspondence, as described below.Comparisons between two sequences are typically performed by comparingthe sequences over a comparison window to identify and compare localregions of sequence similarity. A “comparison window” as used herein,refers to a segment of at least about 20 contiguous positions, usually30 to about 75, 40 to about 50, in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned.

Optimal alignment of sequences for comparison may be conducted using theMegalign program in the Lasergene suite of bioinformatics software(DNASTAR, Inc., Madison, Wis.), using default parameters. This programembodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA80:726-730.

Alternatively, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman (1981)Add. APL. Math 2:482, by the identity alignment algorithm of Needlemanand Wunsch (1970) J. Mol. Biol. 48:443, by the search for similaritymethods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT,BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or byinspection.

One preferred example of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al. (1977)Nucl. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol.215:403-410, respectively. BLAST and BLAST 2.0 can be used, for examplewith the parameters described herein, to determine percent sequenceidentity for the polynucleotides of the invention. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. In one illustrative example,cumulative scores can be calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments, (B) of 50, expectation (E) of 10, M=5, N=4 and a comparisonof both strands.

Preferably, the “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a window of comparison ofat least 20 positions, wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less, usually 5 to 15 percent, or 10 to 12percent, as compared to the reference sequences (which does not compriseadditions or deletions) for optimal alignment of the two sequences. Thepercentage is calculated by determining the number of positions at whichthe identical nucleic acid bases occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the reference sequence (i.e., thewindow size) and multiplying the results by 100 to yield the percentageof sequence identity.

It will be appreciated by those of ordinary skill in the art that, as aresult of the degeneracy of the genetic code, there are many nucleotidesequences that encode a polypeptide as described herein. Some of thesepolynucleotides bear minimal homology to the nucleotide sequence of anynative gene. Nonetheless, polynucleotides that vary due to differencesin codon usage are specifically contemplated by the present invention.Further, alleles of the genes comprising the polynucleotide sequencesprovided herein are within the scope of the present invention. Allelesare endogenous genes that are altered as a result of one or moremutations, such as deletions, additions and/or substitutions ofnucleotides. The resulting mRNA and protein may, but need not, have analtered structure or function. Alleles may be identified using standardtechniques (such as hybridization, amplification and/or databasesequence comparison).

Therefore, in another embodiment of the invention, a mutagenesisapproach, such as site-specific mutagenesis, is employed for thepreparation of immunogenic variants and/or derivatives of thepolypeptides described herein. By this approach, specific modificationsin a polypeptide sequence can be made through mutagenesis of theunderlying polynucleotides that encode them. These techniques provides astraightforward approach to prepare and test sequence variants, forexample, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into thepolynucleotide.

Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Mutations may be employed in aselected polynucleotide sequence to improve, alter, decrease, modify, orotherwise change the properties of the polynucleotide itself, and/oralter the properties, activity, composition, stability, or primarysequence of the encoded polypeptide.

In certain embodiments of the present invention, the inventorscontemplate the mutagenesis of the disclosed polynucleotide sequences toalter one or more properties of the encoded polypeptide, such as theimmunogenicity of a polypeptide vaccine. The techniques of site-specificmutagenesis are well-known in the art, and are widely used to createvariants of both polypeptides and polynucleotides. For example,site-specific mutagenesis is often used to alter a specific portion of aDNA molecule. In such embodiments, a primer comprising typically about14 to about 25 nucleotides or so in length is employed, with about 5 toabout 10 residues on both sides of the junction of the sequence beingaltered.

As will be appreciated by those of skill in the art, site-specificmutagenesis techniques have often employed a phage vector that exists inboth a single stranded and double stranded form. Typical vectors usefulin site-directed mutagenesis include vectors such as the M13 phage.These phage are readily commercially-available and their use isgenerally well-known to those skilled in the art. Double-strandedplasmids are also routinely employed in site directed mutagenesis thateliminates the step of transferring the gene of interest from a plasmidto a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double-stranded vector that includes within itssequence a DNA sequence that encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis provides a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.Specific details regarding these methods and protocols are found in theteachings of Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991;Kuby, 1994; and Maniatis et al., 1982, each incorporated herein byreference, for that purpose.

As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” is intended to refer to a process that involvesthe template-dependent extension of a primer molecule. The term templatedependent process refers to nucleic acid synthesis of an RNA or a DNAmolecule wherein the sequence of the newly synthesized strand of nucleicacid is dictated by the well-known rules of complementary base pairing(see, for example, Watson, 1987). Typically, vector mediatedmethodologies involve the introduction of the nucleic acid fragment intoa DNA or RNA vector, the clonal amplification of the vector, and therecovery of the amplified nucleic acid fragment. Examples of suchmethodologies are provided by U.S. Pat. No. 4,237,224, specificallyincorporated herein by reference in its entirety.

In another approach for the production of polypeptide variants of thepresent invention, recursive sequence recombination, as described inU.S. Pat. No. 5,837,458, may be employed. In this approach, iterativecycles of recombination and screening or selection are performed to“evolve” individual polynucleotide variants of the invention having, forexample, enhanced immunogenic activity.

In other embodiments of the present invention, the polynucleotidesequences provided herein can be advantageously used as probes orprimers for nucleic acid hybridization. As such, it is contemplated thatnucleic acid segments that comprise a sequence region of at least about15 nucleotide long contiguous sequence that has the same sequence as, oris complementary to, a 15 nucleotide long contiguous sequence disclosedherein will find particular utility. Longer contiguous identical orcomplementary sequences, e.g., those of about 20, 30, 40, 50, 100, 200,500, 1000 (including all intermediate lengths) and even up to fulllength sequences will also be of use in certain embodiments.

The ability of such nucleic acid probes to specifically hybridize to asequence of interest will enable them to be of use in detecting thepresence of complementary sequences in a given sample. However, otheruses are also envisioned, such as the use of the sequence informationfor the preparation of mutant species primers, or primers for use inpreparing other genetic constructions.

Polynucleotide molecules having sequence regions consisting ofcontiguous nucleotide stretches of 10-14, 15-20, 30, 50, or even of100-200 nucleotides or so (including intermediate lengths as well),identical or complementary to a polynucleotide sequence disclosedherein, are particularly contemplated as hybridization probes for usein, e.g., Southern and Northern blotting. This would allow a geneproduct, or fragment thereof, to be analyzed, both in diverse cell typesand also in various bacterial cells. The total size of fragment, as wellas the size of the complementary stretch(es), will ultimately depend onthe intended use or application of the particular nucleic acid segment.Smaller fragments will generally find use in hybridization embodiments,wherein the length of the contiguous complementary region may be varied,such as between about 15 and about 100 nucleotides, but largercontiguous complementarity stretches may be used, according to thelength complementary sequences one wishes to detect.

The use of a hybridization probe of about 15-25 nucleotides in lengthallows the formation of a duplex molecule that is both stable andselective. Molecules having contiguous complementary sequences overstretches greater than 15 bases in length are generally preferred,though, in order to increase stability and selectivity of the hybrid,and thereby improve the quality and degree of specific hybrid moleculesobtained. One will generally prefer to design nucleic acid moleculeshaving gene-complementary stretches of 15 to 25 contiguous nucleotides,or even longer where desired.

Hybridization probes may be selected from any portion of any of thesequences disclosed herein. All that is required is to review thesequences set forth herein, or to any continuous portion of thesequences, from about 15-25 nucleotides in length up to and-includingthe full length sequence, that one wishes to utilize as a probe orprimer. The choice of probe and primer sequences may be governed byvarious factors. For example, one may wish to employ primers fromtowards the termini of the total sequence.

Small polynucleotide segments or fragments may be readily prepared by,for example, directly synthesizing the fragment by chemical means, as iscommonly practiced using an automated oligonucleotide synthesizer. Also,fragments may be obtained by application of nucleic acid reproductiontechnology, such as the PCR™ technology of U.S. Pat. No. 4,683,202(incorporated herein by reference), by introducing selected sequencesinto recombinant vectors for recombinant production, and by otherrecombinant DNA techniques generally known to those of skill in the artof molecular biology.

The nucleotide sequences of the invention may be used for their abilityto selectively form duplex molecules with complementary stretches of theentire gene or gene fragments of interest. Depending on the applicationenvisioned, one will typically desire to employ varying conditions ofhybridization to achieve varying degrees of selectivity of probe towardstarget sequence. For applications requiring high selectivity, one willtypically desire to employ relatively stringent conditions to form thehybrids, e.g., one will select relatively low salt and/or hightemperature conditions, such as provided by a salt concentration of fromabout 0.02 M to about 0.15 M salt at temperatures of from about 50° C.to about 70° C. Such selective conditions tolerate little, if any,mismatch between the probe and the template or target strand, and wouldbe particularly suitable for isolating related sequences.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template, less stringent (reduced stringency) hybridizationconditions will typically be needed in order to allow formation of theheteroduplex. In these circumstances, one may desire to employ saltconditions such as those of from about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Cross-hybridizingspecies can thereby be readily identified as positively hybridizingsignals with respect to control hybridizations. In any case, it isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formamide, which serves todestabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

According to another embodiment of the present invention, polynucleotidecompositions comprising antisense oligonucleotides are provided.Antisense oligonucleotides have been demonstrated to be effective andtargeted inhibitors of protein syntheses, and, consequently, provide atherapeutic approach by which a disease can be treated by inhibiting thesynthesis of proteins that contribute to the disease. The efficacy ofantisense oligonucleotides for inhibiting protein synthesis is wellestablished. For example, the synthesis of polygalactauronase and themuscarine type 2 acetylcholine receptor are inhibited by antisenseoligonucleotides directed to their respective mRNA sequences (U.S. Pat.No. 5,739,119 and U.S. Pat. No. 5,759,829). Further, examples ofantisense inhibition have been demonstrated with the nuclear proteincyclin, the multiple drug resistance gene (MDG1), ICAM-1, E-selectin,STK-1, striatal GABA_(A) receptor and human EGF (Jaskulski et al.,Science. Jun. 10, 1988;240(4858):1544-6; Vasanthakumar and Ahmed, CancerCommun. 1989; 1(4):225-32; Peris et al., Brain Res Mol Brain Res. Jun.15, 1998;57(2):310-20; U.S. Pat. No. 5,801,154; U.S. Pat. No. 5,789,573;U.S. Pat. No. 5,718,709 and U.S. Pat. No. 5,610,288). Antisenseconstructs have also been described that inhibit and can be used totreat a variety of abnormal cellular proliferations, e.g. cancer (U.S.Pat. No. 5,747,470; U.S. Pat. No. 5,591,317 and U.S. Pat. No.5,783,683).

Therefore, in certain embodiments, the present invention providesoligonucleotide sequences that comprise all, or a portion of, anysequence that is capable of specifically binding to polynucleotidesequence described herein, or a complement thereof. In one embodiment,the antisense oligonucleotides comprise DNA or derivatives thereof. Inanother embodiment, the oligonucleotides comprise RNA or derivativesthereof. In a third embodiment, the oligonucleotides are modified DNAscomprising a phosphorothioated modified backbone. In a fourthembodiment, the oligonucleotide sequences comprise peptide nucleic acidsor derivatives thereof. In each case, preferred compositions comprise asequence region that is complementary, and more preferablysubstantially-complementary, and even more preferably, completelycomplementary to one or more portions of polynucleotides disclosedherein. Selection of antisense compositions specific for a given genesequence is based upon analysis of the chosen target sequence anddetermination of secondary structure, T_(m), binding energy, andrelative stability. Antisense compositions may be selected based upontheir relative inability to form dimers, hairpins, or other secondarystructures that would reduce or prohibit specific binding to the targetmRNA in a host cell. Highly preferred target regions of the mRNA, arethose which are at or near the AUG translation initiation codon, andthose sequences which are substantially complementary to 5′ regions ofthe mRNA. These secondary structure analyses and target site selectionconsiderations can be performed, for example, using v.4 of the OLIGOprimer analysis software and/or the BLASTN 2.0.5 algorithm software(Altschul et al., Nucleic Acids Res. 1997, 25(17):3389-402).

The use of an antisense delivery method employing a short peptidevector, termed MPG (27 residues), is also contemplated. The MPG peptidecontains a hydrophobic domain derived from the fusion sequence of HIVgp41 and a hydrophilic domain from the nuclear localization sequence ofSV40 T-antigen (Morris et al., Nucleic Acids Res. Jul. 15,1997;25(14):2730-6). It has been demonstrated that several molecules ofthe MPG peptide coat the antisense oligonucleotides and can be deliveredinto cultured mammalian cells in less than 1 hour with relatively highefficiency (90%). Further, the interaction with MPG strongly increasesboth the stability of the oligonucleotide to nuclease and the ability tocross the plasma membrane.

According to another embodiment of the invention, the polynucleotidecompositions described herein are used in the design and preparation ofribozyme molecules for inhibiting expression of the tumor polypeptidesand proteins of the present invention in tumor cells. Ribozymes areRNA-protein complexes that cleave nucleic acids in a site-specificfashion. Ribozymes have specific catalytic domains that possessendonuclease activity (Kim and Cech, Proc Natl Acad Sci USA. 1987December;84(24):8788-92; Forster and Symons, Cell. Apr. 24,1987;49(2):211-20). For example, a large number of ribozymes acceleratephosphoester transfer reactions with a high degree of specificity, oftencleaving only one of several phosphoesters in an oligonucleotidesubstrate (Cech et al., Cell. 1981 December;27(3 Pt 2):487-96; Micheland Westhof, J Mol Biol. Dec. 5, 1990;216(3):585-610; Reinhold-Hurek andShub, Nature. May 14, 1992;357(6374):173-6). This specificity has beenattributed to the requirement that the substrate bind via specificbase-pairing interactions to the internal guide sequence (“IGS”) of theribozyme prior to chemical reaction.

Six basic varieties of naturally-occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin trans (and thus can cleave other RNA molecules) under physiologicalconditions. In general, enzymatic nucleic acids act by first binding toa target RNA. Such binding occurs through the target binding portion ofa enzymatic nucleic acid which is held in close proximity to anenzymatic portion of the molecule that acts to cleave the target RNA.Thus, the enzymatic nucleic acid first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After an enzymatic nucleic acid hasbound and cleaved its RNA target, it is released from that RNA to searchfor another target and can repeatedly bind and cleave new targets.

The enzymatic nature of a ribozyme is advantageous over manytechnologies, such as antisense technology (where a nucleic acidmolecule simply binds to a nucleic acid target to block its translation)since the concentration of ribozyme necessary to affect a therapeutictreatment is lower than that of an antisense oligonucleotide. Thisadvantage reflects the ability of the ribozyme to act enzymatically.Thus, a single ribozyme molecule is able to cleave many molecules oftarget RNA. In addition, the ribozyme is a highly specific inhibitor,with the specificity of inhibition depending not only on the basepairing mechanism of binding to the target RNA, but also on themechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can completely eliminatecatalytic activity of a ribozyme. Similar mismatches in antisensemolecules do not prevent their action (Woolf et al., Proc Natl Acad SciUSA. Aug. 15, 1992;89(16):7305-9). Thus, the specificity of action of aribozyme is greater than that of an antisense oligonucleotide bindingthe same RNA site.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, a hepatitis δ virus, group I intron or RNaseP RNA (inassociation with an RNA guide sequence) or Neurospora VS RNA motif.Examples of hammerhead motifs are described by Rossi et al. NucleicAcids Res. Sep. 11, 1992;20(17):4559-65. Examples of hairpin motifs aredescribed by Hampel et al. (Eur. Pat. Appl. Publ. No. EP 0360257),Hampel and Tritz, Biochemistry Jun. 13, 1989;28(12):4929-33; Hampel etal., Nucleic Acids Res. Jan. 25, 1990;18(2):299-304 and U.S. Pat. No.5,631,359. An example of the hepatitis δ virus motif is described byPerrotta and Been, Biochemistry. Dec. 1, 1992;31(47):11843-52; anexample of the RNaseP motif is described by Guerrier-Takada et al.,Cell. 1983 December;35(3 Pt 2):849-57; Neurospora VS RNA ribozyme motifis described by Collins (Saville and Collins, Cell. May 18,1990;61(4):685-96; Saville and Collins, Proc Natl Acad Sci USA. Oct. 1,1991;88(19):8826-30; Collins and Olive, Biochemistry. Mar. 23,1993;32(11):2795-9); and an example of the Group I intron is describedin (U.S. Pat. No. 4,987,071). All that is important in an enzymaticnucleic acid molecule of this invention is that it has a specificsubstrate binding site which is complementary to one or more of thetarget gene RNA regions, and that it have nucleotide sequences within orsurrounding that substrate binding site which impart an RNA cleavingactivity to the molecule. Thus the ribozyme constructs need not belimited to specific motifs mentioned herein.

Ribozymes may be designed as described in Int. Pat. Appl. Publ. No. WO93/23569 and Int. Pat. Appl. Publ. No. WO 94/02595, each specificallyincorporated herein by reference) and synthesized to be tested in vitroand in vivo, as described. Such ribozymes can also be optimized fordelivery. While specific examples are provided, those in the art willrecognize that equivalent RNA targets in other species can be utilizedwhen necessary.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms, or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases (seee.g., Int. Pat. Appl. Publ. No. WO 92/07065; Int. Pat. Appl. Publ. No.WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl.Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ.No. WO 94/13688, which describe various chemical modifications that canbe made to the sugar moieties of enzymatic RNA molecules), modificationswhich enhance their efficacy in cells, and removal of stem II bases toshorten RNA synthesis times and reduce chemical requirements.

Sullivan et al. (Int. Pat. Appl. Publ. No. WO 94/02595) describes thegeneral methods for delivery of enzymatic RNA molecules. Ribozymes maybe administered to cells by a variety of methods known to those familiarto the art, including, but not restricted to, encapsulation inliposomes, by iontophoresis, or by incorporation into other vehicles,such as hydrogels, cyclodextrins, biodegradable nanocapsules, andbioadhesive microspheres. For some indications, ribozymes may bedirectly delivered ex vivo to cells or tissues with or without theaforementioned vehicles. Alternatively, the RNA/vehicle combination maybe locally delivered by direct inhalation, by direct injection or by useof a catheter, infusion pump or stent. Other routes of delivery include,but are not limited to, intravascular, intramuscular, subcutaneous orjoint injection, aerosol inhalation, oral (tablet or pill form),topical, systemic, ocular, intraperitoneal and/or intrathecal delivery.More detailed descriptions of ribozyme delivery and administration areprovided in Int. Pat. Appl. Publ. No. WO 94/02595 and Int. Pat. Appl.Publ. No. WO 93/23569, each specifically incorporated herein byreference.

Another means of accumulating high concentrations of a ribozyme(s)within cells is to incorporate the ribozyme-encoding sequences into aDNA expression vector. Transcription of the ribozyme sequences aredriven from a promoter for eukaryotic RNA polymerase I (pol I), RNApolymerase II (pol II), or RNA polymerase III (pol III). Transcriptsfrom pol II or pol III promoters will be expressed at high levels in allcells; the levels of a given pol II promoter in a given cell type willdepend on the nature of the gene regulatory sequences (enhancers,silencers, etc.) present nearby. Prokaryotic RNA polymerase promotersmay also be used; providing that the prokaryotic RNA polymerase enzymeis expressed in the appropriate cells Ribozymes expressed from suchpromoters have been shown to function in mammalian cells. Suchtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated vectors), or viral RNA vectors (such as retroviral,semliki forest virus, sindbis virus vectors).

In another embodiment of the invention, peptide nucleic acids (PNAs)compositions are provided. PNA is a DNA mimic in which the nucleobasesare attached to a pseudopeptide backbone (Good and Nielsen, AntisenseNucleic Acid Drug Dev. 1997 7(4) 431-37). PNA is able to be utilized ina number methods that traditionally have used RNA or DNA. Often PNAsequences perform better in techniques than the corresponding RNA or DNAsequences and have utilities that are not inherent to RNA or DNA. Areview of PNA including methods of making, characteristics of, andmethods of using, is provided by Corey (Trends Biotechnol 1997June;15(6):224-9). As such, in certain embodiments, one may prepare PNAsequences that are complementary to one or more portions of the ACE mRNAsequence, and such PNA compositions may be used to regulate, alter,decrease, or reduce the translation of ACE-specific mRNA, and therebyalter the level of ACE activity in a host cell to which such PNAcompositions have been administered.

PNAs have 2-aminoethyl-glycine linkages replacing the normalphosphodiester backbone of DNA (Nielsen et al., Science Dec. 6,1991;254(5037):1497-500; Hanvey et al., Science. Nov. 27,1992;258(5087):1481-5; Hyrup and Nielsen, Bioorg Med Chem. 1996January;4(1):5-23). This chemistry has three important consequences:firstly, in contrast to DNA or phosphorothioate oligonucleotides, PNAsare neutral molecules; secondly, PNAs are achiral, which avoids the needto develop a stereoselective synthesis; and thirdly, PNA synthesis usesstandard Boc or Fmoc protocols for solid-phase peptide synthesis,although other methods, including a modified Merrifield method, havebeen used.

PNA monomers or ready-made oligomers are commercially available fromPerSeptive Biosystems (Framingham, Mass.). PNA syntheses by either Bocor Fmoc protocols are straightforward using manual or automatedprotocols (Norton et al., Bioorg Med Chem. 1995 April;3(4):437-45). Themanual protocol lends itself to the production of chemically modifiedPNAs or the simultaneous synthesis of families of closely related PNAs.

As with peptide synthesis, the success of a particular PNA synthesiswill depend on the properties of the chosen sequence. For example, whilein theory PNAs can incorporate any combination of nucleotide bases, thepresence of adjacent purines can lead to deletions of one or moreresidues in the product. In expectation of this difficulty, it issuggested that, in producing PNAs with adjacent purines, one shouldrepeat the coupling of residues likely to be added inefficiently. Thisshould be followed by the purification of PNAs by reverse-phasehigh-pressure liquid chromatography, providing yields and purity ofproduct similar to those observed during the synthesis of peptides.

Modifications of PNAs for a given application may be accomplished bycoupling amino acids during solid-phase synthesis or by attachingcompounds that contain a carboxylic acid group to the exposed N-terminalamine. Alternatively, PNAs can be modified after synthesis by couplingto an introduced lysine or cysteine. The ease with which PNAs can bemodified facilitates optimization for better solubility or for specificfunctional requirements. Once synthesized, the identity of PNAs andtheir derivatives can be confirmed by mass spectrometry. Several studieshave made and utilized modifications of PNAs (for example, Norton etal., Bioorg Med Chem. 1995 April;3(4):437-45; Petersen et al., J PeptSci. 1995 May-June;1(3):175-83; Orum et al., Biotechniques. 1995September;19(3):472-80; Footer et al., Biochemistry. Aug. 20,1996;35(33):10673-9; Griffith et al., Nucleic Acids Res. Aug. 11,1995;23(15):3003-8; Pardridge et al., Proc Natl Acad Sci USA. Jun. 6,1995;92(12):5592-6; Boffa et al., Proc Natl Acad Sci USA. Mar. 14,1995;92(6):1901-5; Gambacorti-Passerini et al., Blood. Aug. 15,1996;88(4):1411-7; Armitage et al., Proc Natl Acad Sci USA. Nov. 11,1997;94(23):12320-5; Seeger et al., Biotechniques. 1997September;23(3):512-7). U.S. Pat. No. 5,700,922 discusses PNA-DNA-PNAchimeric molecules and their uses in diagnostics, modulating protein inorganisms, and treatment of conditions susceptible to therapeutics.

Methods of characterizing the antisense binding properties of PNAs arediscussed in Rose (Anal Chem. Dec. 15. 1993;65(24):3545-9) and Jensen etal. (Biochemistry. Apr. 22, 1997;36(16):5072-7). Rose uses capillary gelelectrophoresis to determine binding of PNAs to their complementaryoligonucleotide, measuring the relative binding kinetics andstoichiometry. Similar types of measurements were made by Jensen et al.using BIAcore™ technology.

Other applications of PNAs that have been described and will be apparentto the skilled artisan include use in DNA strand invasion, antisenseinhibition, mutational analysis, enhancers of transcription, nucleicacid purification, isolation of transcriptionally active genes, blockingof transcription factor binding, genome cleavage, biosensors, in situhybridization, and the like.

Polynucleotide Identification Characterization and Expression

Polynucleotides compositions of the present invention may be identified,prepared and/or manipulated using any of a variety of well establishedtechniques (see generally, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor,N.Y., 1989, and other like references). For example, a polynucleotidemay be identified, as described in more detail below, by screening amicroarray of cDNAs for tumor-associated expression (i.e., expressionthat is at least two fold greater in a tumor than in normal tissue, asdetermined using a representative assay provided herein). Such screensmay be performed, for example, using the microarray technology ofAffymetrix, Inc. (Santa Clara, Calif.) according to the manufacturer'sinstructions (and essentially as described by Schena et al., Proc. Natl.Acad. Sci. USA 93:10614-10619, 1996 and Heller et al., Proc. Natl. Acad.Sci. USA 94:2150-2155, 1997). Alternatively, polynucleotides may beamplified from cDNA prepared from cells expressing the proteinsdescribed herein, such as tumor cells.

Many template dependent processes ate available to amplify a targetsequences of interest present in a sample. One of the best knownamplification methods is the polymerase chain reaction (PCR™) which isdescribed in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and4,800,159, each of which is incorporated herein by reference in itsentirety. Briefly, in PCR™, two primer sequences are prepared which arecomplementary to regions on opposite complementary strands of the targetsequence. An excess of deoxynucleoside triphosphates is added to areaction mixture along with a DNA polymerase (e.g., Taq polymerase). Ifthe target sequence is present in a sample, the primers will bind to thetarget and the polymerase will cause the primers to be extended alongthe target sequence by adding on nucleotides. By raising and loweringthe temperature of the reaction mixture, the extended primers willdissociate from the target to form reaction products, excess primerswill bind to the target and to the reaction product and the process isrepeated. Preferably reverse transcription and PCR™ amplificationprocedure may be performed in order to quantify the amount of mRNAamplified. Polymerase chain reaction methodologies are well known in theart.

Any of a number of other template dependent processes, many of which arevariations of the PCR™ amplification technique, are readily known andavailable in the art. Illustratively, some such methods include theligase chain reaction (referred to as LCR), described, for example, inEur. Pat. Appl. Publ. No. 320,308 and U.S. Pat. No. 4,883,750; QbetaReplicase, described in PCT Intl. Pat. Appl. Publ. No. PCT/US87/00880;Strand Displacement Amplification (SDA) and Repair Chain Reaction (RCR).Still other amplification methods are described in Great Britain Pat.Appl. No. 2 202 328, and in PCT Intl. Pat. Appl. Publ. No.PCT/US89/01025. Other nucleic acid amplification procedures includetranscription-based amplification systems (TAS) (PCT Intl. Pat. Appl.Publ. No. WO 88/10315), including nucleic acid sequence basedamplification (NASBA) and 3SR. Eur. Pat. Appl. Publ. No. 329,822describes a nucleic acid amplification process involving cyclicallysynthesizing single-stranded RNA (“ssRNA”), ssDNA, and double-strandedDNA (dsDNA). PCT Intl. Pat. Appl. Publ. No. WO 89/06700 describes anucleic acid sequence amplification scheme based on the hybridization ofa promoter/primer sequence to a target single-stranded DNA (“ssDNA”)followed by transcription of many RNA copies of the sequence. Otheramplification methods such as “RACE” (Frohman, 1990), and “one-sidedPCR” (Ohara, 1989) are also well-known to those of skill in the art.

An amplified portion of a polynucleotide of the present invention may beused to isolate a full length gene from a suitable library (e.g., atumor cDNA library) using well known techniques. Within such techniques,a library (cDNA or genomic) is screened using one or more polynucleotideprobes or primers suitable for amplification. Preferably, a library issize-selected to include larger molecules. Random primed libraries mayalso be preferred for identifying 5′ and upstream regions of genes.Genomic libraries are preferred for obtaining introns and extending 5′sequences.

For hybridization techniques, a partial sequence may be labeled (e.g.,by nick-translation or end-labeling with ³²p) using well knowntechniques. A bacterial or bacteriophage library is then generallyscreened by hybridizing filters containing denatured bacterial colonies(or lawns containing phage plaques) with the labeled probe (see Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y., 1989). Hybridizing colonies orplaques are selected and expanded, and the DNA is isolated for furtheranalysis. cDNA clones may be analyzed to determine the amount ofadditional sequence by, for example, PCR using a primer from the partialsequence and a primer from the vector. Restriction maps and partialsequences may be generated to identify one or more overlapping clones.The complete sequence may then be determined using standard techniques,which may involve generating a series of deletion clones. The resultingoverlapping sequences can then assembled into a single contiguoussequence. A full length cDNA molecule can be generated by ligatingsuitable fragments, using well known techniques.

Alternatively, amplification techniques, such as those described above,can be useful for obtaining a full length coding sequence from a partialcDNA sequence. One such amplification technique is inverse PCR (seeTriglia et al., Nucl. Acids Res. 16:8186, 1988), which uses restrictionenzymes to generate a fragment in the known region of the gene. Thefragment is then circularized by intramolecular ligation and used as atemplate for PCR with divergent primers derived from the known region.Within an alternative approach, sequences adjacent to a partial sequencemay be retrieved by amplification with a primer to a linker sequence anda primer specific to a known region. The amplified sequences aretypically subjected to a second round of amplification with the samelinker primer and a second primer specific to the known region. Avariation on this procedure, which employs two primers that initiateextension in opposite directions from the known sequence, is describedin WO 96/38591. Another such technique is known as “rapid amplificationof cDNA ends” or RACE. This technique involves the use of an internalprimer and an external primer, which hybridizes to a polyA region orvector sequence, to identify sequences that are 5′ and 3′ of a knownsequence. Additional techniques include capture PCR (Lagerstrom et al.,PCR Methods Applic. 1:111-19, 1991) and walking PCR (Parker et al.,Nucl. Acids. Res. 19:3055-60, 1991). Other methods employingamplification may also be employed to obtain a full length cDNAsequence.

In certain instances, it is possible to obtain a full length cDNAsequence by analysis of sequences provided in an expressed sequence tag(EST) database, such as that available from GenBank. Searches foroverlapping ESTs may generally be performed using well known programs(e.g., NCBI BLAST searches), and such ESTs may be used to generate acontiguous full length sequence. Full length DNA sequences may also beobtained by analysis of genomic fragments.

In other embodiments of the invention, polynucleotide sequences orfragments thereof which encode polypeptides of the invention, or fusionproteins or functional equivalents thereof, may be used in recombinantDNA molecules to direct expression of a polypeptide in appropriate hostcells. Due to the inherent degeneracy of the genetic code, other DNAsequences that encode substantially the same or a functionallyequivalent amino acid sequence may be produced and these sequences maybe used to clone and express a given polypeptide.

As will be understood by those of skill in the art, it may beadvantageous in some instances to produce polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence.

Moreover, the polynucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterpolypeptide encoding sequences for a variety of reasons, including butnot limited to, alterations which modify the cloning, processing, and/orexpression of the gene product. For example, DNA shuffling by randomfragmentation and PCR reassembly of gene fragments and syntheticoligonucleotides may be used to engineer the nucleotide sequences. Inaddition, site-directed mutagenesis may be used to insert newrestriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, or introduce mutations, and soforth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences may be ligated to a heterologoussequence to encode a fusion protein. For example, to screen peptidelibraries for inhibitors of polypeptide activity, it may be useful toencode a chimeric protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the polypeptide-encoding sequence and theheterologous protein sequence, so that the polypeptide may be cleavedand purified away from the heterologous moiety.

Sequences encoding a desired polypeptide may be synthesized, in whole orin part, using chemical methods well known-in the art (see Caruthers, M.H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al.(1980) Nucl. Acids Res. Symp. Ser. 225-232). Alternatively, the proteinitself may be produced using chemical methods to synthesize the aminoacid sequence of a polypeptide, or a portion thereof. For example,peptide synthesis can be performed using various solid-phase techniques(Roberge, J. Y. et al. (1995) Science 269:202-204) and automatedsynthesis may be achieved, for example, using the ABI 431A PeptideSynthesizer (Perkin Elmer, Palo Alto, Calif.).

A newly synthesized peptide may be substantially purified by preparativehigh performance liquid chromatography (e.g., Creighton, T. (1983)Proteins, Structures and Molecular Principles, WH Freeman and Co., NewYork, N.Y.) or other comparable techniques available in the art. Thecomposition of the synthetic peptides may be confirmed by amino acidanalysis or sequencing (e.g., the Edman degradation procedure).Additionally, the amino acid sequence of a polypeptide, or any partthereof, may be altered during direct synthesis and/or combined usingchemical methods with sequences from other proteins, or any partthereof, to produce a variant polypeptide.

In order to express a desired polypeptide, the nucleotide sequencesencoding the polypeptide, or functional equivalents, may be insertedinto appropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods which are well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding a polypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described, for example, in Sambrook,J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) CurrentProtocols in Molecular Biology, John Wiley & Sons, New York. N.Y.

A variety of expression vector/host systems may be utilized to containand express polynucleotide sequences. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thePBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

In bacterial systems, any of a number of expression vectors may beselected depending upon the use intended for the expressed polypeptide.For example, when large quantities are needed, for example for theinduction of antibodies, vectors which direct high level expression offusion proteins that are readily purified may be used. Such vectorsinclude, but are not limited to, the multifunctional E. coli cloning andexpression vectors such as BLUESCRIPT (Stratagene), in which thesequence encoding the polypeptide of interest may be ligated into thevector in frame with sequences for the amino-terminal Met and thesubsequent 7 residues of beta.-galactosidase so that a hybrid protein isproduced; pIN vectors (Van Heeke, G. and S. M. Schuster (1989) J. Biol.Chem. 264:5503-5509); and the like. pGEX Vectors (Promega, Madison,Wis.) may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems may bedesigned to include heparin, thrombin, or factor XA protease cleavagesites so that the cloned polypeptide of interest can be released fromthe GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding polypeptides may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311.Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196).

An insect system may also be used to express a polypeptide of interest.For example, in one such system, Autographa califomica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. The sequencesencoding the polypeptide may be cloned into a non-essential region ofthe virus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of the polypeptide-encodingsequence will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which the polypeptide of interest may be expressed (Engelhard,E. K. et al. (1994) Proc. Natl. Acad. Sci. 91 :3224-3227).

In mammalian host cells, a number of viral-based expression systems aregenerally available. For example, in cases where an adenovirus is usedas an expression vector, sequences encoding a polypeptide of interestmay be ligated into an adenovirus transcription/translation complexconsisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus which is capable of expressing thepolypeptide in infected host cells (Logan, J. and Shenk, T. (1984) Proc.Natl. Acad. Sci. 81:3655-3659). In addition, transcription enhancers,such as the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding a polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf, D. et al.(1994) Results Probl. Cell Differ. 20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, COS, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is generally preferred. For example, cell lines which stablyexpress a polynucleotide of interest may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover trans formed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1990) Cell 22:817-23) geneswhich can be employed in tk.sup.- or aprt.sup.-cells, respectively.Also, antimetabolite, antibiotic or herbicide resistance can be used asthe basis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosides,neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14); and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,Which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51). Theuse of visible markers has gained popularity with such markers asanthocyanins, beta-glucuronidase and its substrate GUS, and luciferaseand its substrate luciferin, being widely used not only to identifytransformants, but also to quantify the amount of transient or stableprotein expression attributable to a specific vector system (Rhodes, C.A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding apolypeptide is inserted within a marker gene sequence, recombinant cellscontaining sequences can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with apolypeptide-encoding sequence under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells that contain and express a desiredpolynucleotide sequence may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridizaetions and protein bioassayor immunoassay techniques which include, for example, membrane,solution, or chip based technologies for the detection and/orquantification of nucleic acid or protein.

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products, using either polyclonal or monoclonalantibodies specific for the product are known in the art. Examplesinclude enzyme-linked immunosorbent assay (ELISA), radioimmunoassay(RIA), and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on a given polypeptide may be preferred forsome applications, but a competitive binding assay may also be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul. Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides include oligolabeling,nick translation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the sequences, or any portions thereof may becloned into a vector for the production of an mRNA probe. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits. Suitable reporter molecules or labels, which may be used includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with a polynucleotide sequence of interest may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides of theinvention may be designed to contain signal sequences which directsecretion of the encoded polypeptide through a prokaryotic or eukaryoticcell membrane. Other recombinant constructions may be used to joinsequences encoding a polypeptide of interest to nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins. Such purification facilitating domains include, butare not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen. San Diego, Calif.) between the purificationdomain and the encoded polypeptide may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing a polypeptide of interest and a nucleic acidencoding 6 histidine residues preceding a thioredoxin or an enterokinasecleavage site. The histidine residues facilitate purification on IMIAC(immobilized metal ion affinity chromatography) as described in Porath,J. et al. (1992, Prot. Exp. Purif. 3:263-281) while the enterokinasecleavage site provides a means for purifying the desired polypeptidefrom the fusion protein. A discussion of vectors which contain fusionproteins is provided in Kroll, D. J. et al. (1993; DNA Cell Biol.12:441-453).

In addition to recombinant production methods, polypeptides of theinvention, and fragments thereof, may be produced by direct peptidesynthesis using solid-phase techniques (Merrifield J. (1963) J. Am.Chem. Soc. 85:2149-2154). Protein synthesis may be performed usingmanual techniques or by automation. Automated synthesis may be achieved,for example, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer). Alternatively, various fragments may be chemically synthesizedseparately and combined using chemical methods to produce the fulllength molecule.

Antibody Compositions Fragments Thereof and Other Binding Agents

According to another aspect, the present invention further providesbinding agents, such as antibodies and antigen-binding fragmentsthereof, that exhibit immunological binding to a tumor polypeptidedisclosed herein, or to a portion, variant or derivative thereof. Anantibody, or antigen-binding fragment thereof, is said to “specificallybind,” “immunogically bind,” and/or is “immunologically reactive” to apolypeptide of the invention if it reacts at a detectable level (within,for example, an ELISA assay) with the polypeptide, and does not reactdetectably with unrelated polypeptides under similar conditions.

Immunological binding, as used in this context, generally refers to thenon-covalent interactions of the type which occur between animmunoglobulin molecule and an antigen for which the immunoglobulin isspecific. The strength, or affinity of immunological bindinginteractions can be expressed in terms of the dissociation constant(K_(d)) of the interaction, wherein a smaller K_(d) represents a greateraffinity. Immunological binding properties of selected polypeptides canbe quantified using methods well known in the art. One such methodentails measuring the rates of antigen-binding site/antigen complexformation and dissociation, wherein those rates depend on theconcentrations of the complex partners, the affinity of the interaction,and on geometric parameters that equally influence the rate in bothdirections. Thus, both the “on rate constant” (K_(on)) and the “off rateconstant” (K_(off)) can be determined by calculation of theconcentrations and the actual rates of association and dissociation. Theratio of K_(off)/K_(on) enables cancellation of all parameters notrelated to affinity, and is thus equal to the dissociation constantK_(d). See, generally, Davies et al. (1990) Annual Rev. Biochem.59:439-473.

An “antigen-binding site,” or “binding portion” of an antibody refers tothe part of the immunoglobulin molecule that participates in antigenbinding. The antigen binding site is formed by amino acid residues ofthe N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”)chains. Three highly divergent stretches within the V regions of theheavy and light chains are referred to as “hypervariable regions” whichare interposed between more conserved flanking stretches known as“framework regions,” or “FRs”. Thus the term “FR” refers to amino acidsequences which are naturally found between and adjacent tohypervariable regions in immunoglobulins. In an antibody molecule, thethree hypervariable regions of a light chain and the three hypervariableregions of a heavy chain are disposed relative to each other in threedimensional space to form an antigen-binding surface. Theantigen-binding surface is complementary to the three-dimensionalsurface of a bound antigen, and the three hypervariable regions of eachof the heavy and light chains are referred to as“complementarity-determining regions,” or “CDRs.”

Binding agents may be further capable of differentiating betweenpatients with and without a cancer, such as breast cancer, using therepresentative assays provided herein. For example, antibodies or otherbinding agents that bind to a tumor protein will preferably generate asignal indicating the presence of a cancer in at least about 20% ofpatients with the disease, more preferably at least about 30% ofpatients. Alternatively, or in addition, the antibody will generate anegative signal indicating the absence of the disease in at least about90% of individuals without the cancer. To determine whether a bindingagent satisfies this requirement, biological samples (e.g., blood, sera,sputum, urine and/or tumor biopsies) from patients with and without acancer (as determined using standard clinical tests) may be assayed asdescribed herein for the presence of polypeptides that bind to thebinding agent. Preferably, a statistically significant number of sampleswith and without the disease will be assayed. Each binding agent shouldsatisfy the above criteria; however, those of ordinary skill in the artwill recognize that binding agents may be used in combination to improvesensitivity.

Any agent that satisfies the above requirements may be a binding agent.For example, a binding agent may be a ribosome, with or without apeptide component, an RNA molecule or a polypeptide. In a preferredembodiment, a binding agent is an antibody or an antigen-bindingfragment thereof. Antibodies may be prepared by any of a variety oftechniques known to those of ordinary skill in the art. See, e.g.,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988. In general, antibodies can be produced by cell culturetechniques, including the generation of monoclonal antibodies asdescribed herein, or via transfection of antibody genes into suitablebacterial or mammalian cell hosts, in order to allow for the productionof recombinant antibodies. In one technique, an immunogen comprising thepolypeptide is initially injected into any of a wide variety of mammals(e.g., mice, rats, rabbits, sheep or goats). In this step, thepolypeptides of this invention may serve as the immunogen withoutmodification. Alternatively, particularly for relatively shortpolypeptides, a superior immune response may be elicited if thepolypeptide is joined to a carrier protein, such as bovine serum albuminor keyhole limpet hemocyanin. The immunogen is injected into the animalhost, preferably according to a predetermined schedule incorporating oneor more booster immunizations, and the animals are bled periodically.Polyclonal antibodies specific for the polypeptide may then be purifiedfrom such antisera by, for example, affinity chromatography using thepolypeptide coupled to a suitable solid support.

Monoclonal antibodies specific for an antigenic polypeptide of interestmay be prepared, for example, using the technique of Kohler andMilstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto.Briefly, these methods involve the preparation of immortal cell linescapable of producing antibodies having the desired specificity (i.e.,reactivity with the polypeptide of interest). Such cell lines may beproduced, for example, from spleen cells obtained from an animalimmunized as described above. The spleen cells are then immortalized by,for example, fusion with a myeloma cell fusion partner, preferably onethat is syngeneic with the immunized animal. A variety of fusiontechniques may be employed. For example, the spleen cells and myelomacells may be combined with a nonionic detergent for a few minutes andthen plated at low density on a selective medium that supports thegrowth of hybrid cells, but not myeloma cells. A preferred selectiontechnique uses HAT (hypoxanthine, aminopterin, thymidine) selection.After a sufficient time, usually about 1 to 2 weeks, colonies of hybridsare observed. Single colonies are selected and their culturesupernatants tested for binding activity against the polypeptide.Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction. The polypeptides of this invention may beused in the purification process in, for example, an affinitychromatography step.

A number of therapeutically useful molecules are known in the art whichcomprise antigen-binding sites that are capable of exhibitingimmunological binding properties of an antibody molecule. Theproteolytic enzyme papain preferentially cleaves IgG molecules to yieldseveral fragments, two of which (the “F(ab)” fragments) each comprise acovalent heterodimer that includes an intact antigen-binding site. Theenzyme pepsin is able to cleave IgG molecules to provide severalfragments, including the “F(ab′)₂” fragment which comprises bothantigen-binding sites. An “Fv” fragment can be produced by preferentialproteolytic cleavage of an IgM, and on rare occasions IgG or IgAimmunoglobulin molecule. Fv fragments are, however, more commonlyderived using recombinant techniques known in the art. The Fv fragmentincludes a non-covalent V_(H)::V_(L) heterodimer including anantigen-binding site which retains much of the antigen recognition andbinding capabilities of the native antibody molecule. Inbar et al.(1972) Proc. Nat. Acad. Sci. USA 69:2659-2662; Hochman et al. (1976)Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.

A single chain Fv (“sFv”) polypeptide is a covalently linkedV_(H)::V_(L) heterodimer which is expressed from a gene fusion includingV_(H)- and V_(L)-encoding genes linked by a peptide-encoding linker.Huston et al. (1988) Proc. Nat. Acad. Sci. USA 85(16):5879-5883. Anumber of methods have been described to discern chemical structures forconverting the naturally aggregated—but chemically separated—light andheavy polypeptide chains from an antibody V region into an sFv moleculewhich will fold into a three dimensional structure substantially similarto the structure of an antigen-binding site. See, e.g., U.S. Pat. Nos.5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778,to Ladner et al.

Each of the above-described molecules includes a heavy chain and a lightchain CDR set, respectively interposed between a heavy chain and a lightchain FR set which provide support to the CDRS and define the spatialrelationship of the CDRs relative to each other. As used herein, theterm “CDR set” refers to the three hypervariable regions of a heavy orlight chain V region. Proceeding from the N-terminus of a heavy or lightchain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3”respectively. An antigen-binding site, therefore, includes six CDRs,comprising the CDR set from each of a heavy and a light chain V region.A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3) isreferred to herein as a “molecular recognition unit.” Crystallographicanalysis of a number of antigen-antibody complexes has demonstrated thatthe amino acid residues of CDRs form extensive contact with boundantigen, wherein the most extensive antigen contact is with the heavychain CDR3. Thus, the molecular recognition units are primarilyresponsible for the specificity of an antigen-binding site.

As used herein, the term “FR set” refers to the four flanking amino acidsequences which frame the CDRs of a CDR set of a heavy or light chain Vregion. Some FR residues may contact bound antigen; however, FRs areprimarily responsible for folding the V region into the antigen-bindingsite, particularly the FR residues directly adjacent to the CDRS. WithinFRs, certain amino residues and certain structural features are veryhighly conserved. In this regard, all V region sequences contain aninternal disulfide loop of around 90 amino acid residues. When the Vregions fold into a binding-site, the CDRs are displayed as projectingloop motifs which form an antigen-binding surface. It is generallyrecognized that there are conserved structural regions of FRs whichinfluence the folded shape of the CDR loops into certain “canonical”structures—regardless of the precise CDR amino acid sequence. Further,certain FR residues are known to participate in non-covalent interdomaincontacts which stabilize the interaction of the antibody heavy and lightchains.

A number of “humanized” antibody molecules comprising an antigen-bindingsite derived from a non-human immunoglobulin have been described,including chimeric antibodies having rodent V regions and theirassociated CDRs fused to human constant domains (Winter et al. (1991)Nature 349:293-299; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA86:4220-4224; Shaw et al. (1987) J Immunol. 138:4534-4538; and Brown etal. (1987) Cancer Res. 47:3577-3583), rodent CDRs grafted into a humansupporting FR prior to fusion with an appropriate human antibodyconstant domain (Riechmann et al. (1988) Nature 332:323-327; Verhoeyenet al. (1988) Science 239:1534-1536; and Jones et al. (1986) Nature321:522-525), and rodent CDRs supported by recombinantly veneered rodentFRs (European Patent Publication No. 519,596, published Dec. 23, 1992).These “humanized” molecules are designed to minimize unwantedimmunological response toward rodent antihuman antibody molecules whichlimits the duration and effectiveness of therapeutic applications ofthose moieties in human recipients.

As used herein, the terms “veneered FRs” and “recombinantly veneeredFRs” refer to the selective replacement of FR residues from, e.g., arodent heavy or light chain V region, with human FR residues in order toprovide a xenogeneic molecule comprising an antigen-binding site whichretains substantially all of the native FR polypeptide foldingstructure. Veneering techniques are based on the understanding that theligand binding characteristics of an antigen-binding site are determinedprimarily by the structure and relative disposition of the heavy andlight chain CDR sets within the antigen-binding surface. Davies et al.(1990) Ann. Rev. Biochem. 59:439-473. Thus, antigen binding specificitycan be preserved in a humanized antibody only wherein the CDRstructures, their interaction with each other, and their interactionwith the rest of the V region domains are carefully maintained. By usingveneering techniques, exterior (e.g., solvent-accessible) FR residueswhich are readily encountered by the immune system are selectivelyreplaced with human residues to provide a hybrid molecule that compriseseither a weakly immunogenic, or substantially non-immunogenic veneeredsurface.

The process of veneering makes use of the available sequence data forhuman antibody variable domains compiled by Kabat et al., in Sequencesof Proteins of Immunological Interest, 4th ed., (U.S. Dept. of Healthand Human Services, U.S. Government Printing Office, 1987), updates tothe Kabat database, and other accessible U.S. and foreign databases(both nucleic acid and protein). Solvent accessibilities of V regionamino acids can be deduced from the known three-dimensional structurefor human and murine antibody fragments. There are two general steps inveneering a murine antigen-binding site. Initially, the FRs of thevariable domains of an antibody molecule of interest are compared withcorresponding FR sequences of human variable domains obtained from theabove-identified sources. The most homologous human V regions are thencompared residue by residue to corresponding murine amino acids. Theresidues in the murine FR which differ from the human counterpart arereplaced by the residues present in the human moiety using recombinanttechniques well known in the art. Residue switching is only carried outwith moieties which are at least partially exposed (solvent accessible),and care is exercised in the replacement of amino acid residues whichmay have a significant effect on the tertiary structure of V regiondomains, such as proline, glycine and charged amino acids.

In this manner, the resultant “veneered” murine antigen-binding sitesare thus designed to retain the murine CDR residues, the residuessubstantially adjacent to the CDRs, the residues identified as buried ormostly buried (solvent inaccessible), the residues believed toparticipate in non-covalent (e.g., electrostatic and hydrophobic)contacts between heavy and light chain domains, and the residues fromconserved structural regions of the FRs which are believed to influencethe “canonical” tertiary structures of the CDR loops. These designcriteria are then used to prepare recombinant nucleotide sequences whichcombine the CDRs of both the heavy and light chain of a murineantigen-binding site into human-appearing FRs that can be used totransfect mammalian cells for the expression of recombinant humanantibodies which exhibit the antigen specificity of the murine antibodymolecule.

In another embodiment of the invention, monoclonal antibodies of thepresent invention may be coupled to one or more therapeutic agents.Suitable agents in this regard include radionuclides, differentiationinducers, drugs, toxins, and derivatives thereof. Preferredradionuclides include ⁹⁰Y, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ²¹¹At, and²¹²Bi. Preferred drugs include methotrexate, and pyrimidine and purineanalogs. Preferred differentiation inducers include phorbol esters andbutyric acid. Preferred toxins include ricin, abrin, diptheria toxin,cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, andpokeweed antiviral protein.

A therapeutic agent may be coupled (e.g., covalently bonded) to asuitable monoclonal antibody either directly or indirectly (e.g., via alinker group). A direct reaction between an agent and an antibody ispossible when each possesses a substituent capable of reacting with theother. For example, a nucleophilic group, such as an amino or sulfhydrylgroup, on one may be capable of reacting with a carbonyl-containinggroup, such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide) on the other.

Alternatively, it may be desirable to couple a therapeutic agent and anantibody via a linker group. A linker group can function as a spacer todistance an antibody from an agent in order to avoid interference withbinding capabilities. A linker group can also serve to increase thechemical reactivity of a substituent on an agent or an antibody, andthus increase the coupling efficiency. An increase in chemicalreactivity may also facilitate the use of agents, or functional groupson agents, which otherwise would not be possible.

It will be evident to those skilled in the art that a variety ofbifunctional or polyfunctional reagents, both homo- andhetero-functional (such as those described in the catalog of the PierceChemical Co., Rockford, Ill.), may be employed as the linker group.Coupling may be effected, for example, through amino groups, carboxylgroups, sulfhydryl groups or oxidized carbohydrate residues. There arenumerous references describing such methodology, e.g., U.S. Pat. No.4,671,958,to Rodwell et al.

Where a therapeutic agent is more potent when free from the antibodyportion of the immunoconjugates of the present invention, it may bedesirable to use a linker group which is cleavable during or uponinternalization into a cell. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof an agent from these linker groups include cleavage by reduction of adisulfide bond (e.g., U.S. Pat. No. 4,489,710, to Spitler), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014, toSenter et al.), by hydrolysis of derivatized amino acid side chains(e.g., U.S. Pat. No.,4,638,045, to Kohn et al.), by serumcomplement-mediated hydrolysis (e.g., U.S. Pat. No. 4,671,958, toRodwell et al.), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789, to Blattler et al.).

It may be desirable to couple more than one agent to an antibody. In oneembodiment, multiple molecules of an agent are coupled to one antibodymolecule. In another embodiment, more than one type of agent may becoupled to one antibody. Regardless of the particular embodiment,immunoconjugates with more than one agent may be prepared in a varietyof ways. For example, more than one agent may be coupled directly to anantibody molecule, or linkers that provide multiple sites for attachmentcan be used. Alternatively, a carrier can be used.

A carrier may bear the agents in a variety of ways, including covalentbonding either directly or via a linker group. Suitable carriers includeproteins such as albumins (e.g., U.S. Pat. No. 4,507,234, to Kato etal.), peptides and polysaccharides such as aminodextran (e.g., U.S. Pat.No. 4,699,784, to Shih et al.). A carrier may also bear an agent bynoncovalent bonding or by encapsulation, such as within a liposomevesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088). Carriersspecific for radionuclide agents include radiohalogenated smallmolecules and chelating compounds. For example, U.S. Pat. No. 4,735,792discloses representative radiohalogenated small molecules and theirsynthesis. A radionuclide chelate may be formed from chelating compoundsthat include those containing nitrogen and sulfur atoms as the donoratoms for binding the metal, or metal oxide, radionuclide. For example,U.S. Pat. No. 4,673,562, to Davison et al. discloses representativechelating compounds and their synthesis.

T Cell Compositions

The present invention, in another aspect, provides T cells specific fora tumor polypeptide disclosed herein, or for a variant or derivativethereof. Such cells may generally be prepared in vitro or ex vivo, usingstandard procedures. For example, T cells may be isolated from bonemarrow, peripheral blood, or a fraction of bone marrow or peripheralblood of a patient, using a commercially available cell separationsystem, such as the Isolex™ System, available from Nexell Therapeutics,Inc. (Irvine, Calif.; see also U.S. Pat. No. 5,240,856; U.S. Pat. No.5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243). Alternatively, Tcells may be derived from related or unrelated humans, non-humanmammals, cell lines or cultures.

T cells may be stimulated with a polypeptide, polynucleotide encoding apolypeptide and/or an antigen presenting cell (APC) that expresses sucha polypeptide. Such stimulation is performed under conditions and for atime sufficient to permit the generation of T cells that are specificfor the polypeptide of interest. Preferably, a tumor polypeptide orpolynucleotide of the invention is present within a delivery vehicle,such as a microsphere, to facilitate the generation of specific T cells.

T cells are considered to be specific for a polypeptide of the presentinvention if the T cells specifically proliferate, secrete cytokines orkill target cells coated with the polypeptide or expressing a geneencoding the polypeptide. T cell specificity may be evaluated using anyof a variety of standard techniques. For example, within a chromiumrelease assay or proliferation assay, a stimulation index of more thantwo fold increase in lysis and/or proliferation, compared to negativecontrols, indicates T cell specificity. Such assays may be performed,for example, as described in Chen et al., Cancer Res. 54:1065-1070,1994. Alternatively, detection of the proliferation of T cells may beaccomplished by a variety of known techniques. For example, T cellproliferation can be detected by measuring an increased rate of DNAsynthesis (e.g., by pulse-labeling cultures of T cells with tritiatedthymidine and measuring the amount of tritiated thymidine incorporatedinto DNA). Contact with a tumor polypeptide (100 ng/ml−100 μg/ml,preferably 200 ng/ml−25 ∥g/ml) for 3-7 days will typically result in atleast a two fold increase in proliferation of the T cells. Contact asdescribed above for 2-3 hours should result in activation of the Tcells, as measured using standard cytokine assays in which a two foldincrease in the level of cytokine release (e.g., TNF or IFN-γ) isindicative of T cell activation (see Coligan et al., Current Protocolsin Immunology, vol. 1, Wiley Interscience (Greene 1998)). T cells thathave been activated in response to a tumor polypeptide, polynucleotideor polypeptide-expressing APC may be CD4⁺ and/or CD8⁺. Tumorpolypeptide-specific T cells may be expanded using standard techniques.Within preferred embodiments, the T cells are derived from a patient, arelated donor or an unrelated donor, and are administered to the patientfollowing stimulation and expansion.

For therapeutic purposes, CD4⁺ or CD8⁺ T cells that proliferate inresponse to a tumor polypeptide, polynucleotide or APC can be expandedin number either in vitro or in vivo. Proliferation of such T cells invitro may be accomplished in a variety of ways. For example, the T cellscan be re-exposed to a tumor polypeptide, or a short peptidecorresponding to an immunogenic portion of such a polypeptide, with orwithout the addition of T cell growth factors, such as interleukin-2,and/or stimulator cells that synthesize a tumor polypeptide.Alternatively, one or more T cells that proliferate in the presence ofthe tumor polypeptide can be expanded in number by cloning. Methods forcloning cells are well known in the art, and include limiting dilution.

Pharmaceutical Compositions

In additional embodiments, the present invention concerns formulation ofone or more of the polynucleotide, polypeptide, T-cell and/or antibodycompositions disclosed herein in pharmaceutically-acceptable carriersfor administration to a cell or an animal, either alone, or incombination with one or more other modalities of therapy.

It will be understood that, if desired, a composition as disclosedherein may be administered in combination with other agents as well,such as, e.g., other proteins or polypeptides or variouspharmaceutically-active agents. In fact there is virtually no limit toother components that may also be included, given that the additionalagents do not cause a significant adverse effect upon contact with thetarget cells or host tissues. The compositions may thus be deliveredalong with various other agents as required in the particular instance.Such compositions may be purified from host cells or other biologicalsources, or alternatively may be chemically synthesized as describedherein. Likewise, such compositions may further comprise substituted orderivatized RNA or DNA compositions.

Therefore, in another aspect of the present invention, pharmaceuticalcompositions are provided comprising one or more of the polynucleotide,polypeptide, antibody, and/or T-cell compositions described herein incombination with a physiologically acceptable carrier. In certainpreferred embodiments, the pharmaceutical compositions of the inventioncomprise immunogenic polynucleotide and/or polypeptide compositions ofthe invention for use in prophylactic and theraputic vaccineapplications. Vaccine preparation is generally described in, forexample, M. F. Powell and M. J. Newman, eds., “Vaccine Design (thesubunit and adjuvant approach),” Plenum Press (NY, 1995). Generally,such compositions will comprise-one or more polynucleotide and/orpolypeptide compositions of the present invention in combination withone or more immunostimulants.

It will be apparent that any of the pharmaceutical compositionsdescribed herein can contain pharmaceutically acceptable salts of thepolynucleotides and polypeptides of the invention. Such salts can beprepared, for example, from pharmaceutically acceptable non-toxic bases,including organic bases (e.g., salts of primary, secondary and tertiaryamines and basic amino acids) and inorganic bases (e.g., sodium,potassium, lithium, ammonium, calcium and magnesium salts).

In another embodiment, illustrative immunogenic compositions, e.g.,vaccine compositions, of the present invention comprise DNA encoding oneor more of the polypeptides as described above, such that thepolypeptide is generated in situ. As noted above, the polynucleotide maybe administered within any of a variety of delivery systems known tothose of ordinary skill in the art. Indeed, numerous gene deliverytechniques are well known in the art, such as those described byRolland, Crit. Rev. Therap. Drug Carrier Systems 15:143-198, 1998, andreferences cited therein. Appropriate polynucleotide expression systemswill, of course, contain the necessary regulatory DNA regulatorysequences for expression in a patient (such as a suitable promoter andterminating signal). Alternatively, bacterial delivery systems mayinvolve the administration of a bacterium (such asBacillus-Calmette-Guerrin) that expresses an immunogenic portion of thepolypeptide on its cell surface or secretes such an epitope.

Therefore, in certain embodiments, polynucleotides encoding immunogenicpolypeptides described herein are introduced into suitable mammalianhost cells for expression using any of a number of known viral-basedsystems. In one illustrative embodiment, retroviruses provide aconvenient and effective platform for gene delivery systems. A selectednucleotide sequence encoding a polypeptide of the present invention canbe inserted into a vector and packaged in retroviral particles usingtechniques known in the art. The recombinant virus can then be isolatedand delivered to a subject. A number of illustrative retroviral systemshave been described (e.g., U.S. Pat. No. 5,219,740; Miller and Rosman(1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993)Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin(1993) Cur. Opin. Genet. Develop. 3:102-109.

In addition, a number of illustrative adenovirus-based systems have alsobeen described. Unlike retroviruses which integrate into the hostgenome, adenoviruses persist extrachromosomally thus minimizing therisks associated with insertional mutagenesis (Haj-Ahmad and Graham(1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921;Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al.(1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58;Berkner, K. L. (1988) BioTechniques 6:616-629; and Rich et al. (1993)Human Gene Therapy 4:461-476).

Various adeno-associated virus (AAV) vector systems have also beendeveloped for polynucleotide delivery. AAV vectors can be readilyconstructed using techniques well known in the art. See, e.g., U.S. Pat.Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070and WO 93/03769; Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996;Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press);Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539;Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shellingand Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp.Med. 179:1867-1875.

Additional viral vectors useful for delivering the polynucleotidesencoding polypeptides of the present invention by gene transfer includethose derived from the pox family of viruses, such as vaccinia virus andavian poxvirus. By way of example, vaccinia virus recombinantsexpressing the novel molecules can be constructed as follows. The DNAencoding a polypeptide is first inserted into an appropriate vector sothat it is adjacent to a vaccinia promoter and flanking vaccinia DNAsequences, such as the sequence encoding thymidine kinase (TK). Thisvector is then used to transfect cells which are simultaneously infectedwith vaccinia. Homologous recombination serves to insert the vacciniapromoter plus the gene encoding the polypeptide of interest into theviral genome. The resulting TK.sup.(−) recombinant can be selected byculturing the cells in the presence of 5-bromodeoxyuridine and pickingviral plaques resistant thereto.

A vaccinia-based infection/transfection system can be conveniently usedto provide for inducible, transient expression or coexpression of one ormore polypeptides described herein in host cells of an organism. In thisparticular system, cells are first infected in vitro with a vacciniavirus recombinant that encodes the bacteriophage T7 RNA polymerase. Thispolymerase displays exquisite specificity in that it only transcribestemplates bearing T7 promoters. Following infection, cells aretransfected with the polynucleotide or polynucleotides of interest,driven by a T7 promoter. The polymerase expressed in the cytoplasm fromthe vaccinia virus recombinant transcribes the transfected DNA into RNAwhich is then translated into polypeptide by the host translationalmachinery. The method provides for high level, transient, cytoplasmicproduction of large quantities of RNA and its translation products. See,e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990)87:6743-6747; Fuerst et al. Proc. Natl. Acad. Sci. USA (1986)83:8122-8126.

Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses,can also be used to deliver the coding sequences of interest.Recombinant avipox viruses, expressing immunogens from mammalianpathogens, are known to confer protective immunity when administered tonon-avian species. The use of an Avipox vector is particularly desirablein human and other mammalian species since members of the Avipox genuscan only productively replicate in susceptible avian species andtherefore are not infective in mammalian cells. Methods for producingrecombinant Avipoxviruses are known in the art and employ geneticrecombination, as described above with respect to the production ofvaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.

Any of a number of alphavirus vectors can also be used for delivery ofpolynucleotide compositions of the present invention, such as thosevectors described in U.S. Pat. Nos. 5,843,723; 6,015,686; 6,008,035 and6,015,694. Certain vectors based on Venezuelan Equine Encephalitis (VEE)can also be used, illustrative examples of which can be found in U.S.Pat. Nos. 5,505,947 and 5,643,576.

Moreover, molecular conjugate vectors, such as the adenovirus chimericvectors described in Michael et al. J. Biol. Chem. (1993) 268:6866-6869and Wagner et al. Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, canalso be used for gene delivery under the invention.

Additional illustrative information on these and other known viral-baseddelivery systems can be found, for example, in Fisher-Hoch et al., Proc.Natl. Acad. Sci. USA 86:317-321, 1989; Flexner et al., Ann. N.Y Acad.Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-21, 1990; U.S. Pat.Nos. 4,603,112, 4,769,330, and 5,017,487; WO 89/01973; U.S. Pat. No.4,777,127; GB 2,200,651; EP 0,345,242; WO 91/02805; Berkner,Biotechniques 6:616-627, 1988; Rosenfeld et al., Science 252:431-434,1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219, 1994;Kass-Eisler et al., Proc. Natl. Acad Sci. USA 90:11498-11502, 1993;Guzman et al., Circulation 88:2838-2848, 1993; and Guzman et al., Cir.Res. 73:1202-1207, 1993.

In certain embodiments, a polynucleotide may be integrated into thegenome of a target cell. This integration may be in the specificlocation and orientation via homologous recombination (gene replacement)or it may be integrated in a random, non-specific location (geneaugmentation). In yet further embodiments, the polynucleotide may bestably maintained in the cell as a separate, episomal segment of DNA.Such polynucleotide segments or “episomes” encode sequences sufficientto permit maintenance and replication independent of or insynchronization with the host cell cycle. The manner in which theexpression construct is delivered to a cell and where in the cell thepolynucleotide remains is dependent on the type of expression constructemployed.

In another embodiment of the invention, a polynucleotide isadministered/delivered as “naked” DNA, for example as described in Ulmeret al., Science 259:1745-1749, 1993 and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto biodegradable beads, which are efficiently transported intothe cells.

In still another embodiment, a composition of the present invention canbe delivered via a particle bombardment approach, many of which havebeen described. In one illustrative example, gas-driven particleacceleration can be achieved with devices such as those manufactured byPowderject Pharmaceuticals PLC (Oxford, UK) and Powderject Vaccines Inc.(Madison, Wis.), some examples of which are described in U.S. Pat. Nos.5,846,796; 6,010,478; 5,865;196; 5,584,807; and EP Patent No. 0500 799.This approach offers a needle-free delivery approach wherein a drypowder formulation of microscopic particles, such as polynucleotide orpolypeptide particles, are accelerated to high speed within a helium gasjet generated by a hand held device, propelling the particles into atarget tissue of interest.

In a related embodiment, other devices and methods that may be usefulfor gas-driven needle-less injection of compositions of the presentinvention include those provided by Bioject, Inc. (Portland, Oreg.),some examples of which are described in U.S. Pat. Nos. 4,790,824;5,064,413; 5,312,335; 5,383,851; 5,399,163; 5,520,639 and 5,993,412.

According to another embodiment, the pharmaceutical compositionsdescribed herein will comprise one or more immunostimulants in additionto the immunogenic polynucleotide, polypeptide, antibody, T-cell and/orAPC compositions of this invention. An immunostimulant refers toessentially any substance that enhances or potentiates an immuneresponse (antibody and/or cell-mediated) to an exogenous antigen. Onepreferred type of immunostimulant comprises an adjuvant. Many adjuvantscontain a substance designed to protect -the antigen from rapidcatabolism, such as aluminum hydroxide or mineral oil, and a stimulatorof immune responses, such as lipid A, Bortadella pertussis orMycobacterium tuberculosis derived proteins. Certain adjuvants arecommercially available as, for example, Freund's Incomplete Adjuvant andComplete Adjuvant (Difco Laboratories, Detroit, Mich.); Merck Adjuvant65 (Merck and Company, Inc., Rahway, N.J.); AS-2 (SmithKline Beecham,Philadelphia, Pa.); aluminum salts such as aluminum hydroxide gel (alum)or aluminum phosphate; salts of calcium, iron or zinc; an insolublesuspension of acylated tyrosine; acylated sugars; cationically oranionically derivatized polysaccharides; polyphosphazenes; biodegradablemicrospheres; monophosphoryl lipid A and quil A. Cytokines, such asGM-CSF, interleukin-2, -7, -12, and other like growth factors, may alsobe used as adjuvants.

Within certain embodiments of the invention, the adjuvant composition ispreferably one that induces an immune response predominantly of the Th1type. High levels of Th1-type cytokines (e.g., IFN-γ, TNFα, IL-2 andIL-12) tend to favor the induction of cell mediated immune responses toan administered antigen. In contrast, high levels of Th2-type cytokines(e.g., IL-4, IL-5, IL-6 and IL-10) tend to favor the induction ofhumoral immune responses. Following application of a vaccine as providedherein, a patient will support an immune response that includes Th1- andTh2-type responses. Within a preferred embodiment, in which a responseis predominantly Th1-type, the level of Th1-type cytokines will increaseto a greater extent than the level of Th2-type cytokines. The levels ofthese cytokines may be readily assessed using standard assays. For areview of the families of cytokines, see Mosmann and Coffman, Ann. Rev.Immunol. 7:145-173, 1989.

Certain preferred adjuvants for eliciting a predominantly Th1-typeresponse include, for example, a combination of monophosphoryl lipid A,preferably 3-de-O-acylated monophosphoryl lipid A, together with analuminum salt. MPL® adjuvants are available from Corixa Corporation(Seattle, Wash.; see, for example, U.S. Pat. Nos. 4,436,727; 4,877,611;4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which theCpG dinucleotide is unmethylated) also induce a predominantly Th1response. Such oligonucleotides are well known and are described, forexample, in WO 96/02555, WO 99/33488 and U.S. Pat. Nos. 6,008,200 and5,856,462. Immunostimulatory DNA sequences are also described, forexample, by Sato et al., Science 273:352, 1996. Another preferredadjuvant comprises a saponin, such as Quil A, or derivatives thereof,including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham,Mass.); Escin; Digitonin; or Gypsophila or Chenopodium quinoa saponins.Other preferred formulations include more than one saponin in theadjuvant combinations of the present invention, for example combinationsof at least two of the following group comprising QS21, QS7, Quil A,β-escin, or digitonin.

Alternatively the saponin formulations may be combined with vaccinevehicles composed of chitosan or other polycationic polymers,polylactide and polylactide-co-glycolide particles, poly-N-acetylglucosamine-based polymer matrix, particles composed of polysaccharidesor chemically modified polysaccharides, liposomes and lipid-basedparticles, particles composed of glycerol monoesters, etc. The saponinsmay also be formulated in the presence of cholesterol to formparticulate structures such as liposomes or ISCOMs. Furthermore, thesaponins may be formulated together with a polyoxyethylene ether orester, in either a non-particulate solution or suspension, or in aparticulate structure such as a paucilamelar liposome or ISCOM. Thesaponins may also be formulated with excipients such as Carbopol^(R) toincrease viscosity, or may be formulated in a dry powder form with apowder excipient such as lactose.

In one preferred embodiment, the adjuvant system includes thecombination of a monophosphoryl lipid A and a saponin derivative, suchas the combination of QS21 and 3D-MPL® adjuvant, as described in WO94/00153, or a less reactogenic composition where the QS21 is quenchedwith cholesterol, as described in WO 96/33739. Other preferredformulations comprise an oil-in-water emulsion and tocopherol. Anotherparticularly preferred adjuvant formulation employing QS21, 3D-MPL®adjuvant and tocopherol in an oil-in-water emulsion is described in WO95/17210.

Another enhanced adjuvant system involves the combination of aCpG-containing oligonucleotide and a saponin derivative particularly thecombination of CpG and QS21 is disclosed in WO 00/09159. Preferably theformulation additionally comprises an oil in water emulsion andtocopherol.

Additional illustrative adjuvants f6r use in the pharmaceuticalcompositions of the invention include Montanide ISA 720 (Seppic,France), SAF (Chiron, Calif., United States), ISCOMS (CSL), MF-59(Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4,available from SmithKline Beecham, Rixensart, Belgium), Detox(Enhanzyn®) (Corixa, Hamilton, Mont.), RC-529 (Corixa, Hamilton, Mont.)and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as thosedescribed in pending U.S. patent application Ser. Nos. 08/853,826 and09/074,720, the disclosures of which are incorporated herein byreference in their entireties, and polyoxyethylene ether adjuvants suchas those described in WO 99/52549A1.

Other preferred adjuvants include adjuvant molecules of the generalformulaHO(CH₂CH₂O)_(n)-A-R,   (I):wherein, n is 1-50, A is a bond or —C(O)—, R is C₁₋₅₀ alkyl or PhenylC₁₋₅₀ alkyl.

One embodiment of the present invention consists of a vaccineformulation comprising a polyoxyethylene ether of general formula (I),wherein n is between 1 and 50, preferably 4-24, most preferably 9; the Rcomponent is C₁₋₅₀, preferably C₄-C₂₀ alkyl and most preferably C₁₂alkyl, and A is a bond. The concentration of the polyoxyethylene ethersshould be in the range 0.1-20%, preferably from 0.1-10%, and mostpreferably in the range 0.1-1%. Preferred polyoxyethylene ethers areselected from the following group: polyoxyethylene-9-lauryl ether,polyoxyethylene-9-steoryl ether, polyoxyethylene-8-steoryl ether,polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, andpolyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such aspolyoxyethylene lauryl ether are described in the Merck index (12^(th)edition: entry 7717). These adjuvant molecules are described in WO99/52549.

The polyoxyethylene ether according to the general formula (I) abovemay, if desired, be combined with another adjuvant. For example, apreferred adjuvant combination is preferably with CpG as described inthe pending UK patent application GB 9820956.2.

According to another embodiment of this invention, an immunogeniccomposition described herein is delivered to a host via antigenpresenting cells (APCs), such as dendritic cells, macrophages, B cells,monocytes and other cells that may be engineered to be efficient APCs.Such cells may, but need not, be genetically modified to increase thecapacity for presenting the antigen, to improve activation and/ormaintenance of the T cell response, to have anti-tumor effects per seand/or to be immunologically compatible with the receiver (i.e., matchedHLA haplotype). APCs may generally be isolated from any of a variety ofbiological fluids and organs, including tumor and peritumoral tissues,and may be autologous, allogeneic, syngeneic or xenogeneic cells.

Certain preferred embodiments of the present invention use dendriticcells or progenitors thereof as antigen-presenting cells. Dendriticcells are highly potent APCs (Banchereau and Steinman, Nature392:245-251, 1998) and have been shown to be effective as aphysiological adjuvant for eliciting prophylactic or therapeuticantitumor immunity (see Timmerman and Levy, Ann. Rev. Med. 50:507-529,1999). In general, dendritic cells may be identified based on theirtypical shape (stellate in situ, with marked cytoplasmic processes(dendrites) visible in vitro), their ability to take up, process andpresent antigens with high efficiency and their ability to activatenaive T cell responses. Dendritic cells may, of course, be engineered toexpress specific cell-surface receptors or ligands that are not commonlyfound on dendritic cells in vivo or ex vivo, and such modified dendriticcells are contemplated by the present invention. As an alternative todendritic cells, secreted vesicles antigen-loaded dendritic cells(called exosomes) may be used within a vaccine (see Zitvogel et al.,Nature Med. 4:594-600, 1998).

Dendritic cells and progenitors may be obtained from peripheral blood,bone marrow, tumor-infiltrating cells, peritumoral tissues-infiltratingcells, lymph nodes, spleen, skin, umbilical cord blood or any othersuitable tissue or fluid. For example, dendritic cells may bedifferentiated ex vivo by adding a combination of cytokines such asGM-CSF, IL-4, IL-13 and/or TNFα to cultures of monocytes harvested fromperipheral blood. Alternatively, CD34 positive cells harvested fromperipheral blood, umbilical cord blood or bone marrow may bedifferentiated into dendritic cells by adding to the culture mediumcombinations of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and/orother compound(s) that induce differentiation, maturation andproliferation of dendritic cells.

Dendritic cells are conveniently categorized as “immature” and “mature”cells, which allows a simple way to discriminate between two wellcharacterized phenotypes. However, this nomenclature should not beconstrued to exclude all possible intermediate stages ofdifferentiation. Immature dendritic cells are characterized as APC witha high capacity for antigen uptake and processing, which correlates withthe high expression of Fcγ receptor and mannose receptor. The maturephenotype is typically characterized by a lower expression of thesemarkers, but a high expression of cell surface molecules responsible forT cell activation such as class I and class II MHC, adhesion molecules(e.g., CD54 and CD11) and costimulatory molecules (e.g., CD40, CD80,CD86 and 4-1BB).

APCs may generally be transfected with a polynucleotide of the invention(or portion or other variant thereof) such that the encoded polypeptide,or an immunogenic portion thereof, is expressed on the cell surface.Such transfection may take place ex vivo, and a pharmaceuticalcomposition comprising such transfected cells may then be used fortherapeutic purposes, as described herein. Alternatively, a genedelivery vehicle that targets a dendritic or other antigen presentingcell may be administered to a patient, resulting in transfection thatoccurs in vivo. In vivo and ex vivo transfection of dendritic cells, forexample, may generally be performed using any methods known in the art,such as those described in WO 97/24447, or the gene gun approachdescribed by Mahvi et al., Immunology and cell Biology 75:456-460, 1997.Antigen loading of dendritic cells may be achieved by incubatingdendritic cells or progenitor cells with the tumor polypeptide, DNA(naked or within a plasmid vector) or RNA; or with antigen-expressingrecombinant bacterium or viruses (e.g., vaccinia, fowlpox, adenovirus orlentivirus vectors). Prior to loading, the polypeptide may be covalentlyconjugated to an immunological partner that provides T cell help (e.g.,a carrier molecule). Alternatively, a dendritic cell may be pulsed witha non-conjugated immunological partner, separately or in the presence ofthe polypeptide.

While any suitable carrier known to those of ordinary skill in the artmay be employed in the pharmaceutical compositions of this invention,the type of carrier will typically vary depending on the mode ofadministration. Compositions of the present invention may be formulatedfor any appropriate manner of administration, including for example,topical, oral, nasal, mucosal, intravenous, intracranial,intraperitoneal, subcutaneous and intramuscular administration.

Carriers for use within such pharmaceutical compositions arebiocompatible, and may also be biodegradable. In certain embodiments,the formulation preferably provides a relatively constant level ofactive component release. In other embodiments, however, a more rapidrate of release immediately upon administration may be desired. Theformulation of such compositions is well within the level of ordinaryskill in the art using known techniques. Illustrative carriers useful inthis regard include microparticles of poly(lactide-co-glycolide),polyacrylate, latex, starch, cellulose, dextran and the like. Otherillustrative delayed-release carriers include supramolecular biovectors,which comprise a non-liquid hydrophilic core (e.g., a cross-linkedpolysaccharide or oligosaccharide) and, optionally, an external layercomprising an amphiphilic compound, such as a phospholipid (see e.g.,U.S. Pat. No. 5,151,254 and PCT applications WO 94/20078, WO/94/23701and WO 96/06638). The amount of active compound contained within asustained release formulation depends upon the site of implantation, therate and expected duration of release and the nature of the condition tobe treated or prevented.

In another illustrative embodiment, biodegradable microspheres (e.g.,polylactate polyglycolate) are employed as carriers for the compositionsof this invention. Suitable biodegradable microspheres are disclosed,for example, in U.S. Pat. Nos. 4,897,268; 5,075,109; 5,928,647;5,811,128; 5,820,883; 5,853,763; 5,814,344, 5,407,609 and 5,942,252.Modified hepatitis B core protein carrier systems. such as described inWO/99 40934, and references cited therein, will also be useful for manyapplications. Another illustrative carrier/delivery system employs acarrier comprising particulate-protein complexes, such as thosedescribed in U.S. Pat. No. 5,928,647, which are capable of inducing aclass I-restricted cytotoxic T lymphocyte responses in a host.

The pharmaceutical compositions of the invention will often furthercomprise one or more buffers (e.g., neutral buffered saline or phosphatebuffered saline), carbohydrates (e.g., glucose, mannose, sucrose ordextrans), mannitol, proteins, polypeptides or amino acids such asglycine, antioxidants, bacteriostats, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide), solutes that renderthe formulation isotonic, hypotonic or weakly hypertonic with the bloodof a recipient, suspending agents, thickening agents and/orpreservatives. Alternatively, compositions of the present invention maybe formulated as a lyophilizate.

The pharmaceutical compositions described herein may be presented inunit-dose or multi-dose containers, such as sealed ampoules or vials.Such containers are typically sealed in such a way to preserve thesterility and stability of the formulation until use. In general,formulations may be stored as suspensions, solutions or emulsions inoily or aqueous vehicles. Alternatively, a pharmaceutical compositionmay be stored in a freeze-dried condition requiring only the addition ofa sterile liquid carrier immediately prior to use.

The development of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens, including e.g., oral, parenteral, intravenous, intranasal, andintramuscular administration and formulation, is well known in the art,some of which are briefly discussed below for general purposes ofillustration.

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to an animal. As such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

The active compounds may even be incorporated with excipients and usedin the form of ingestible tablets, buccal tables, troches, capsules,elixirs, suspensions, syrups, wafers, and the like (see, for example,Mathiowitz et al., Nature Mar. 27, 1997;386(6623):410-4; Hwang et al.,Crit Rev Ther Drug Carrier Syst 1998;15(3):243-84; U.S. Pat. No.5,641,515; U.S. Pat. No. 5,580,579 and U.S. Pat. No. 5,792,451).Tablets, troches, pills, capsules and the like may also contain any of avariety of additional components, for example, a binder, such as gumtragacanth, acacia, cornstarch, or gelatin; excipients, such asdicalcium phosphate; a disintegrating agent, such as corn starch, potatostarch, alginic acid and the like; a lubricant, such as magnesiumstearate; and a sweetening agent, such as sucrose, lactose or saccharinmay be added or a flavoring agent, such as peppermint, oil ofwintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar, or both.Of course, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compounds may be incorporated intosustained-release preparation and formulations.

Typically, these formulations will contain at least about 0.1% of theactive compound or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 60% or 70% or more of the weight or volume ofthe total formulation. Naturally, the amount of active compound(s) ineach therapeutically useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. Alternatively, the active ingredientmay be incorporated into an oral solution such as one containing sodiumborate, glycerin and potassium bicarbonate, or dispersed in adentifrice, or added in a therapeutically-effective amount to acomposition that may include water, binders, abrasives, flavoringagents, foaming agents, and humectants. Alternatively the compositionsmay be fashioned into a tablet or solution form that may be placed underthe tongue or otherwise dissolved in the mouth.

In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,intravenously, intramuscularly, or even intraperitoneally. Suchapproaches are well known to the skilled artisan, some of which arefurther described, for example, in U.S. Pat. No. 5,543,158; U.S. Pat.No. 5,641,515 and U.S. Pat. No. 5,399,363. In certain embodiments,solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations generally will contain a preservative to prevent the growthof microorganisms.

Illustrative pharmaceutical forms suitable for injectable use includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (for example, see U.S. Pat. No. 5,466,468). In all cases theform must be sterile and must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, and/or vegetable oils.Proper fluidity may be maintained, for example, by the use of a coating,such as lecithin, by the maintenance of the required particle size inthe case of dispersion and/or by the use of surfactants. The preventionof the action of microorganisms can be facilitated by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

In one embodiment, for parenteral administration in an aqueous solution,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. Moreover, for human administration, preparationswill of course preferably meet sterility, pyrogenicity, and the generalsafety and purity standards as required by FDA Office of Biologicsstandards.

In another embodiment of the invention, the compositions disclosedherein may be formulated in a neutral or salt form. Illustrativepharmaceutically-acceptable salts include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective.

The carriers can further comprise any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions. The phrase “pharmaceutically-acceptable” refersto molecular entities and compositions that do not produce an allergicor similar untoward reaction when administered to a human.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering genes, nucleic acids, and peptidecompositions directly to the lungs via nasal aerosol sprays has beendescribed, e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212.Likewise, the delivery of drugs using intranasal microparticle resins(Takenaga et al., J Controlled Release Mar. 2, 1998;52(1-2):81-7) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871) are alsowell-known in the pharmaceutical arts. Likewise, illustrativetransmucosal drug delivery in the form of a polytetrafluoroetheylenesupport-matrix is described in U.S. Pat. No. 5,780,045.

In certain embodiments, liposomes, nanocapsules, microparticles, lipidparticles, vesicles, and the like, are used for the introduction of thecompositions of the present invention into suitable hostcells/organisms. In particular, the compositions of the presentinvention may be formulated for delivery either encapsulated in a lipidparticle, a liposome, a vesicle, a nanosphere, or a nanoparticle or thelike. Alternatively, compositions of the present invention can be bound,either covalently or non-covalently, to the surface of such carriervehicles.

The formation and use of liposome and liposome-like preparations aspotential drug carriers is generally known to those of skill in the art(see for example, Lasic, Trends Biotechnol 1998 July;16(7):307-21;Takakura, Nippon Rinsho 1998 March;56(3):691-5; Chandran et al., IndianJ Exp Biol. 1997 August;35(8):801-9; Margalit, Crit Rev Ther DrugCarrier Syst. 1995;12(2-3):233-61; U.S. Pat. No. 5,567,434; U.S. Pat.No. 5,552,157; U.S. Pat. No. 5,565,213; U.S. Pat. No. 5,738,868 and U.S.Pat. No. 5,795,587, each specifically incorporated herein by referencein its entirety).

Liposomes have been used successfully with a number of cell types thatare normally difficult to transfect by other procedures, including Tcell suspensions, primary hepatocyte cultures and PC 12 cells (Renneisenet al., J Biol Chem. Sep. 25, 1990;265(27):16337-42; Muller et al., DNACell Biol. 1990 April;9(3):221-9). In addition, liposomes are free ofthe DNA length constraints that are typical of viral-based deliverysystems. Liposomes have been used effectively to introduce genes,various drugs, radiotherapeutic agents, enzymes, viruses, transcriptionfactors, allosteric effectors and the like, into a variety of culturedcell lines and animals. Furthermore, he use of liposomes does not appearto be associated with autoimmune responses or unacceptable toxicityafter systemic delivery.

In certain embodiments, liposomes are formed from phospholipids that aredispersed in an aqueous medium and spontaneously form multilamellarconcentric bilayer vesicles (also termed multilamellar vesicles (MLVs).

Alternatively, in other embodiments, the invention provides forpharmaceutically-acceptable nanocapsule formulations of the compositionsof the present invention. Nanocapsules can generally entrap compounds ina stable and reproducible way (see, for example, Quintanar-Guerrero etal., Drug Dev Ind Pharm. 1998 December;24(12):1113-28). To avoid sideeffects due to intracellular polymeric overloading, such ultrafineparticles (sized around 0.1. μm) may be designed using polymers able tobe degraded in vivo. Such particles can be made as described, forexample, by Couvreur et al., Crit Rev Ther Drug Carrier Syst.1988;5(1):1-20; zur Muhlen et al., Eur J Pharm Biopharm. 1998March;45(2):149-55; Zambaux et al. J Controlled Release. Jan. 2,1998;50(1-3):31-40; and U.S. Pat. No. 5,145,684.

Cancer Therapeutic Methods

In further aspects of the present invention, the pharmaceuticalcompositions described herein may be used for the treatment of cancer,particularly for the immunotherapy of breast cancer. Within suchmethods, the pharmaceutical compositions described herein areadministered to a patient, typically a warm-blooded animal, preferably ahuman. A patient may or may not be afflicted with cancer. Accordingly,the above pharmaceutical compositions may be used to prevent thedevelopment of a cancer or to treat a patient afflicted with a cancer.Pharmaceutical compositions and vaccines may be administered eitherprior to or following surgical removal of primary tumors and/ortreatment such as administration of radiotherapy or conventionalchemotherapeutic drugs. As discussed above, administration of thepharmaceutical compositions may be by any suitable method, includingadministration by intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal, intradermal, anal, vaginal, topical and oralroutes.

Within certain embodiments, immunotherapy may be active immunotherapy,in which treatment relies on the in vivo stimulation of the endogenoushost immune system to react against tumors with the administration ofimmune response-modifying agents (such as polypeptides andpolynucleotides as provided herein).

Within other embodiments, immunotherapy may be passive immunotherapy, inwhich treatment involves the delivery of agents with establishedtumor-immune reactivity (such as effector cells or antibodies) that candirectly or indirectly mediate antitumor effects and does notnecessarily depend on an intact host immune system. Examples of effectorcells include T cells as discussed above, T lymphocytes (such as CD8⁺cytotoxic T lymphocytes and CD4⁺ T-helper tumor-infiltratinglymphocytes), killer cells (such as Natural Killer cells andlymphokine-activated killer cells), B cells and antigen-presenting cells(such as dendritic cells and macrophages) expressing a polypeptideprovided herein. T cell receptors and antibody receptors specific forthe polypeptides recited herein may be cloned, expressed and transferredinto other vectors or effector cells for adoptive immunotherapy. Thepolypeptides provided herein may also be used to generate antibodies oranti-idiotypic antibodies (as described above and in U.S. Pat. No.4,918,164) for passive immunotherapy.

Effector cells may generally be obtained in sufficient quantities foradoptive immunotherapy by growth in vitro, as described herein. Cultureconditions for expanding single antigen-specific effector cells toseveral billion in number with retention of antigen recognition in vivoare well known in the art. Such in vitro culture conditions typicallyuse intermittent stimulation with antigen, often in the presence ofcytokines (such as IL-2) and non-dividing feeder cells. As noted above,immunoreactive polypeptides as provided herein may be used to rapidlyexpand antigen-specific T cell cultures in order to generate asufficient number of cells for immunotherapy. In particular,antigen-presenting cells, such as dendritic, macrophage, monocyte,fibroblast and/or B cells, may be pulsed with immunoreactivepolypeptides or transfected with one or more polynucleotides usingstandard techniques well known in the art. For example,antigen-presenting cells can be transfected with a polynucleotide havinga promoter appropriate for increasing expression in a recombinant virusor other expression system. Cultured effector cells for use in therapymust be able to grow and distribute widely, and to survive long term invivo. Studies have shown that cultured effector cells can be induced togrow in vivo and to survive long term in substantial numbers by repeatedstimulation with antigen supplemented with IL-2 (see, for example,Cheever et al., Immunological Reviews 157:177, 1997).

Alternatively, a vector expressing a polypeptide recited herein may beintroduced into antigen presenting cells taken from a patient andclonally propagated ex vivo for transplant back into the same patient.Transfected cells may be reintroduced into the patient using any meansknown in the art, preferably in sterile form by intravenous,intracavitary, intraperitoneal or intratumor administration.

Routes and frequency of administration of the therapeutic compositionsdescribed herein, as well as dosage, will vary from individual toindividual, and may be readily established using standard techniques. Ingeneral, the pharmaceutical compositions and vaccines may beadministered by injection (e.g., intracutaneous, intramuscular,intravenous or subcutaneous), intranasally (e.g., by aspiration) ororally. Preferably, between 1 and 10 doses may be administered over a 52week period. Preferably, 6 doses are administered, at intervals of 1month, and booster vaccinations may be given periodically thereafter.Alternate protocols may be appropriate for individual patients. Asuitable dose is an amount of a compound that, when administered asdescribed above, is capable of promoting an anti-tumor immune response,and is at least 10-50% above the basal (i.e., untreated) level. Suchresponse can be monitored by measuring the anti-tumor antibodies in apatient or by vaccine-dependent generation of cytolytic effector cellscapable of killing the patient's tumor cells in vitro. Such vaccinesshould also be capable of causing an immune response that leads to animproved clinical outcome (e.g., more frequent remissions, complete orpartial or longer disease-free survival) in vaccinated patient ascompared to non-vaccinated patients. In general, for pharmaceuticalcompositions and vaccines comprising one or more polypeptides, theamount of each polypeptide present in a dose ranges from about 25 μg to5 mg per kg of host. Suitable dose sizes will vary with the size of thepatient, but will typically range from about 0.1 mL to about 5 mL.

In general, an appropriate dosage and treatment regimen provides theactive compound(s) in an amount sufficient to provide therapeutic and/orprophylactic benefit. Such a response can be monitored by establishingan improved clinical outcome (e.g., more frequent remissions, completeor partial, or longer disease-free survival) in treated patients ascompared to non-treated patients. Increases in preexisting immuneresponses to a tumor protein generally correlate with an improvedclinical outcome. Such immune responses may generally be evaluated usingstandard proliferation, cytotoxicity or cytokine assays, which may beperformed using samples obtained from a patient before and aftertreatment.

Cancer Detection and Diagnostic Compositions, Methods and Kits

In general, a cancer may be detected in a patient based on the presenceof one or more breast tumor proteins and/or polynucleotides encodingsuch proteins in a biological sample (for example, blood, sera, sputumurine and/or tumor biopsies) obtained from the patient. In other words,such proteins may be used as markers to indicate the presence or absenceof a cancer such as breast cancer. In addition, such proteins may beuseful for the detection of other cancers. The binding agents providedherein generally permit detection of the level of antigen that binds tothe agent in the biological sample. Polynucleotide primers and probesmay be used to detect the level of mRNA encoding a tumor protein, whichis also indicative of the presence or absence of a cancer. In general, abreast tumor sequence should be present at a level that is at leastthree fold higher in tumor tissue than in normal tissue

There are a variety of assay formats known to those of ordinary skill inthe art for using a binding agent to detect polypeptide markers in asample. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988. In general, the presence or absenceof a cancer in a patient may be determined by (a) contacting abiological sample obtained from a patient with a binding agent; (b)detecting in the sample a level of polypeptide that binds to the bindingagent; and (c) comparing the level of polypeptide with a predeterminedcut-off value.

In a preferred embodiment, the assay involves the use of binding agentimmobilized on a solid support to bind to and remove the polypeptidefrom the remainder of the sample. The bound polypeptide may then bedetected using a detection reagent that contains a reporter group andspecifically binds to the binding agent/polypeptide complex. Suchdetection reagents may comprise, for example, a binding agent thatspecifically binds to the polypeptide or an antibody or other agent thatspecifically binds to the binding agent, such as an anti-immunoglobulin,protein G, protein A or a lectin. Alternatively, a competitive assay maybe utilized, in which a polypeptide is labeled with a reporter group andallowed to bind to the immobilized binding agent after incubation of thebinding agent with the sample. The extent to which components of thesample inhibit the binding of the labeled polypeptide to the bindingagent is indicative of the reactivity of the sample with the immobilizedbinding agent. Suitable polypeptides for use within such assays includefull length breast tumor proteins and polypeptide portions thereof towhich the binding agent binds, as described above.

The solid support may be any material known to those of ordinary skillin the art to which the tumor protein may be attached. For example, thesolid support may be a test well in a microtiter plate or anitrocellulose or other suitable membrane. Alternatively, the supportmay be a bead or disc, such as glass, fiberglass, latex or a plasticmaterial such as polystyrene or polyvinylchloride. The support may alsobe a magnetic particle or a fiber optic sensor, such as those disclosed,for example, in U.S. Pat. No. 5,359,681. The binding agent may beimmobilized on the solid support using a variety of techniques known tothose of skill in the art, which are amply described in the patent andscientific literature. In the context of the present invention, the term“immobilization” refers to both noncovalent association, such asadsorption, and covalent attachment (which may be a direct linkagebetween the agent and functional groups on the support or may be alinkage by way of a cross-linking agent). Immobilization by adsorptionto a well in a microtiter plate or to a membrane is preferred. In suchcases, adsorption may be achieved by contacting the binding agent, in asuitable buffer, with the solid support for a suitable amount of time.The contact time varies with temperature, but is typically between about1 hour and about 1 day. In general, contacting a well of a plasticmicrotiter plate (such as polystyrene or polyvinylchloride) with anamount of binding agent ranging from about 10 ng to about 10 μg, andpreferably about 100 ng to about 1 μg, is sufficient to immobilize anadequate amount of binding agent.

Covalent attachment of binding agent to a solid support may generally beachieved by first reacting the support with a bifunctional reagent thatwill react with both the support and a functional group, such as ahydroxyl or amino group, on the binding agent. For example, the bindingagent may be covalently attached to supports having an appropriatepolymer coating using benzoquinone or by condensation of an aldehydegroup on the support with an amine and an active hydrogen on the bindingpartner (see, e.g., Pierce Immunotechnology Catalog and Handbook, 1991,at A12-A13).

In certain embodiments, the assay is a two-antibody sandwich assay. Thisassay may be performed by first contacting an antibody that has beenimmobilized on a solid support, commonly the well of a microtiter plate;with the sample, such that polypeptides within the sample are allowed tobind to the immobilized antibody. Unbound sample is then removed fromthe immobilized polypeptide-antibody complexes and a detection reagent(preferably a second antibody capable of binding to a different site onthe polypeptide) containing a reporter group is added. The amount ofdetection reagent that remains bound to the solid support is thendetermined using a method appropriate for the specific reporter group.

More specifically, once the antibody is immobilized on the support asdescribed above, the remaining protein binding sites on the support aretypically blocked. Any suitable blocking agent known to those ofordinary skill in the art, such as bovine serum albumin or Tween 20™(Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody is thenincubated with the sample, and polypeptide is allowed to bind to theantibody. The sample may be diluted with a suitable diluent, such asphosphate-buffered saline (PBS) prior to incubation. In general, anappropriate contact time (i.e., incubation time) is a period of timethat is sufficient to detect the presence of polypeptide within a sampleobtained from an individual with breast cancer. Preferably, the contacttime is sufficient to achieve a level of binding that is at least about95% of that achieved at equilibrium between bound and unboundpolypeptide. Those of ordinary skill in the art will recognize that thetime necessary to achieve equilibrium may be readily determined byassaying the level of binding that occurs over a period of time. At roomtemperature, an incubation time of about 30 minutes is generallysufficient.

Unbound sample may then be removed by washing the solid support with anappropriate buffer, such as PBS containing 0.1% Tween 20™. The secondantibody, which contains a reporter group, may then be added to thesolid support. Preferred reporter groups include those groups recitedabove.

The detection reagent is then incubated with the immobilizedantibody-polypeptide complex for an amount of time sufficient to detectthe bound polypeptide. An appropriate amount of time may generally bedetermined by assaying the level of binding that occurs over a period oftime. Unbound detection reagent is then removed and bound detectionreagent is detected using the reporter group. The method employed fordetecting the reporter group depends upon the nature of the reportergroup. For radioactive groups, scintillation counting orautoradiographic methods are generally appropriate. Spectroscopicmethods may be used to detect dyes, luminescent groups and fluorescentgroups. Biotin may be detected using avidin, coupled to a differentreporter group (commonly a radioactive or fluorescent group or anenzyme). Enzyme reporter groups may generally be detected by theaddition of substrate (generally for a specific period of time),followed by spectroscopic or other analysis of the reaction products.

To determine the presence or absence of a cancer, such as breast cancer,the signal detected from the reporter group that remains bound to thesolid support is generally compared to a signal that corresponds to apredetermined cut-off value. In one preferred embodiment, the cut-offvalue for the detection of a cancer is the average mean signal obtainedwhen the immobilized antibody is incubated with samples from patientswithout the cancer. In general, a sample generating a signal that isthree standard deviations above the predetermined cut-off value isconsidered positive for the cancer. In an alternate preferredembodiment, the cut-off value is determined using a Receiver OperatorCurve, according to the method of Sackett et al., Clinical Epidemiology:A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p.106-7. Briefly, in this embodiment, the cut-off value may be determinedfrom a plot of pairs of true positive rates (i.e., sensitivity) andfalse positive rates (100%-specificity) that correspond to each possiblecut-off value for the diagnostic test result. The cut-off value on theplot that is the closest to the upper left-hand corner (i.e., the valuethat encloses the largest area) is the most accurate cut-off value, anda sample generating a signal that is higher than the cut-off valuedetermined by this method may be considered positive. Alternatively, thecut-off value may be shifted to the left along the plot, to minimize thefalse positive rate, or to the right, to minimize the false negativerate. In general, a sample generating a signal that is higher than thecut-off value determined by this method is considered positive for acancer.

In a related embodiment, the assay is performed in a flow-through orstrip test format, wherein the binding agent is immobilized on amembrane, such as nitrocellulose. In the flow-through test, polypeptideswithin the sample bind to the immobilized binding agent as the samplepasses through the membrane. A second, labeled binding agent then bindsto the binding agent-polypeptide complex as a solution containing thesecond binding agent flows through the membrane. The detection of boundsecond binding agent may then be performed as described above. In thestrip test format, one end of the membrane to which binding agent isbound is immersed in a solution containing the sample. The samplemigrates along the membrane through a region containing second bindingagent and to the area of immobilized binding agent. Concentration ofsecond binding agent at the area of immobilized antibody indicates thepresence of a cancer. Typically, the concentration of second bindingagent at that site generates a pattern, such as a line, that can be readvisually. The absence of such a pattern indicates a negative result. Ingeneral, the amount of binding agent immobilized on the membrane isselected to generate a visually discernible pattern when the biologicalsample contains a level of polypeptide that would be sufficient togenerate a positive signal in the two-antibody sandwich assay, in theformat discussed above. Preferred binding agents for use in such assaysare antibodies and antigen-binding fragments thereof. Preferably, theamount of antibody immobilized on the membrane ranges from about 25 ngto about 1 μg, and more preferably from about 50 ng to about 500 ng.Such tests can typically be performed with a very small amount ofbiological sample.

Of course, numerous other assay protocols exist that are suitable foruse with the tumor proteins or binding agents of the present invention.The above descriptions are intended to be exemplary only. For example,it will be apparent to those of ordinary skill in the art that the aboveprotocols may be readily modified to use tumor polypeptides to detectantibodies that bind to such polypeptides in a biological sample. Thedetection of such tumor protein specific antibodies may correlate withthe presence of a cancer.

A cancer may also, or alternatively, be detected based on the presenceof T cells that specifically react with a tumor protein in a biologicalsample. Within certain methods, a biological sample comprising CD4⁺and/or CD8⁺ T cells isolated from a patient is incubated with a tumorpolypeptide, a polynucleotide encoding such a polypeptide and/or an APCthat expresses at least an immunogenic portion of such a polypeptide,and the presence or absence of specific activation of the T cells isdetected. Suitable biological samples include, but are not limited to,isolated T cells. For example, T cells may be isolated from a patient byroutine-techniques (such as by Ficoll/Hypaque density gradientcentrifugation of peripheral blood lymphocytes). T cells may beincubated in vitro for 2-9 days (typically 4 days) at 37° C. withpolypeptide (e.g., 5-25 μg/ml). It may be desirable to incubate anotheraliquot of a T cell sample in the absence of tumor polypeptide to serveas a control. For CD4⁺ T cells, activation is preferably detected byevaluating proliferation of the T cells. For CD8⁺ T cells, activation ispreferably detected by evaluating cytolytic activity. A level ofproliferation that is at least two fold greater and/or a level ofcytolytic activity that is at least 20% greater than in disease-freepatients indicates the presence of a cancer in the patient.

As noted above, a cancer may also, or alternatively, be detected basedon the level of mRNA encoding a tumor protein in a biological sample.For example, at least two oligonucleotide primers may be employed in apolymerase chain reaction (PCR) based assay to amplify a portion of atumor cDNA derived from a biological sample, wherein at least one of theoligonucleotide primers is specific for (i.e., hybridizes to) apolynucleotide encoding the tumor protein. The amplified cDNA is thenseparated and detected using techniques well known in the art, such asgel electrophoresis. Similarly, oligonucleotide probes that specificallyhybridize to a polynucleotide encoding a tumor protein may be used in ahybridization assay to detect the presence of polynucleotide encodingthe tumor protein in a biological sample.

To permit hybridization under assay conditions, oligonucleotide primersand probes should comprise an oligonucleotide sequence that has at leastabout 60%, preferably at least about 75% and more preferably at leastabout 90%, identity to a portion of a polynucleotide encoding a tumorprotein of the invention that is at least 10 nucleotides, and preferablyat least 20 nucleotides, in length. Preferably, oligonucleotide primersand/or probes hybridize to a polynucleotide encoding a polypeptidedescribed herein under moderately stringent conditions, as definedabove. Oligonucleotide primers and/or probes which may be usefullyemployed in the diagnostic methods described herein preferably are atleast 10-40 nucleotides in length. In a preferred embodiment, theoligonucleotide primers comprise at least 10 contiguous nucleotides,more preferably at least 15 contiguous nucleotides, of a DNA moleculehaving a sequence as disclosed herein. Techniques for both PCR basedassays and hybridization assays are well known in the art (see, forexample, Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263,1987; Erlich ed., PCR Technology, Stockton Press, NY, 1989).

One preferred assay employs RT-PCR, in which PCR is applied inconjunction with reverse transcription. Typically, RNA is extracted froma biological sample, such as biopsy tissue, and is reverse transcribedto produce cDNA molecules. PCR amplification using at least one specificprimer generates a cDNA molecule, which may be separated and visualizedusing, for example, gel electrophoresis. Amplification may be performedon biological samples taken from a test patient and from an individualwho is not afflicted with a cancer. The amplification reaction may beperformed on several dilutions of cDNA spanning two orders of magnitude.A two-fold or greater increase in expression in several dilutions of thetest patient sample as compared to the same dilutions of thenon-cancerous sample is typically considered positive.

In another embodiment, the compositions described herein may be used asmarkers for the progression of cancer. In this embodiment, assays asdescribed above for the diagnosis of a cancer may be performed overtime, and the change in the level of reactive polypeptide(s) orpolynucleotide(s) evaluated. For example, the assays may be performedevery 24-72 hours for a period of 6 months to 1 year, and thereafterperformed as needed. In general, a cancer is progressing in thosepatients in whom the level of polypeptide or polynucleotide detectedincreases over time. In contrast, the cancer is not progressing when thelevel of reactive polypeptide or polynucleotide either remains constantor decreases with time.

Certain in vivo diagnostic assays may be performed directly on a tumor.One such assay involves contacting tumor cells with a binding agent. Thebound binding agent may then be detected directly or indirectly via areporter group. Such binding agents may also be used in histologicalapplications. Alternatively, polynucleotide probes may be used withinsuch applications.

As noted above, to improve sensitivity, multiple tumor protein markersmay be assayed within a given sample. It will be apparent that bindingagents specific for different proteins provided herein may be combinedwithin a single assay. Further, multiple primers or probes may be usedconcurrently. The selection of tumor protein markers may be based onroutine experiments to determine combinations that results in optimalsensitivity. In addition, or alternatively, assays for tumor proteinsprovided herein may be combined with assays for other known tumorantigens.

The present invention further provides kits for use within any of theabove diagnostic methods. Such kits typically comprise two or morecomponents necessary for performing a diagnostic assay. Components maybe compounds, reagents, containers and/or equipment. For example, onecontainer within a kit may contain a monoclonal antibody or fragmentthereof that specifically binds to a tumor protein. Such antibodies orfragments may be provided attached to a support material, as describedabove. One or more additional containers may enclose elements, such asreagents or buffers, to be used in the assay. Such kits may also, oralternatively, contain a detection reagent as described above thatcontains a reporter group suitable for direct or indirect detection ofantibody binding.

Alternatively, a kit may be designed to detect the level of mRNAencoding a tumor protein in a biological sample. Such kits generallycomprise at least one oligonucleotide probe or primer, as describedabove, that hybridizes to a polynucleotide encoding a tumor protein.Such an oligonucleotide may be used, for example, within a PCR orhybridization assay. Additional components that may be present withinsuch kits include a second oligonucleotide and/or a diagnostic reagentor container to facilitate the detection of a polynucleotide encoding atumor protein.

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLE 1 Isolation and Characterization of breast Tumor Polypeptides

This Example describes the isolation of breast tumor polypeptides from abreast tumor cDNA library.

A cDNA subtraction library containing cDNA from breast tumor subtractedwith normal breast cDNA was constructed as follows. Total RNA wasextracted from primary tissues using Trizol reagent (Gibco BRL LifeTechnologies, Gaithersburg, Md.) as described by the manufacturer. ThepolyA+RNA was purified using an oligo(dT) cellulose column according tostandard protocols. First strand cDNA was synthesized using the primersupplied in a Clontech PCR-Select cDNA Subtraction Kit (Clontech, PaloAlto, Calif.). The driver DNA consisted of cDNAs from two normal breasttissues with the tester cDNA being from three primary breast tumors.Double-stranded cDNA was synthesized for both tester and driver, anddigested with a combination of endonucleases (MluI, MscI, PvuII, SalIand StuI) which recognize six base pairs DNA. This modificationincreased the average cDNA size dramatically compared with cDNAsgenerated according to the protocol of Clontech (Palo Alto, Calif.). Thedigested tester cDNAs were ligated to two different adaptors and thesubtraction was performed according to Clontech's protocol. Thesubtracted cDNAs were subjected to two rounds of PCR amplification,following the manufacturer's protocol. The resulting PCR products weresubcloned into the TA cloning vector, pCRII (Invitrogen, San Diego,Calif.) and transformed into ElectroMax E. coli DH10B cells (Gibco BRLLife, Technologies) by electroporation. DNA was isolated fromindependent clones and sequenced using a Perkin Elmer/Applied BiosystemsDivision (Foster City, Calif.) Automated Sequencer Model 373A.

Sixty-three distinct cDNA clones were found in the subtracted breasttumor-specific cDNA library. The determined one strand (5′ or 3′) cDNAsequences for the clones are provided in SEQ ID NO:1-61, 72 and 73,respectively. Comparison of these cDNA sequences with known sequences inthe gene bank using the EMBL and GenBank databases (Release 97) revealedno significant homologies to the sequences provided in SEQ ID NO:14, 21,22, 27, 29, 30, 32, 38, 44, 45, 53, 57, 72 and 73. The sequences of SEQID NO: 1, 3, 16, 17, 34, 48, 60 and 61 were found to represent knownhuman genes. The sequences of SEQ ID NO:2, 4, 23, 39 and 50 were foundto show some similarity to previously identified non-human genes. Theremaining clones (SEQ ID NO:5-13, 15, 18-20, 24-26, 28, 31, 33, 35-37,40-43,-46, 47, 49, 51, 52, 54-56, 58 and 59) were found to show at leastsome degree of homology to previously identified expressed sequence tags(ESTs).

Further studies resulted in the isolation of the full-length cDNAsequence for the clone of SEQ ID NO:57 (referred to as B718P). Bycomputer analysis, the full-length sequence was found to contain aputative transmembrane domain at amino acids 137-158. The full-lengthcDNA sequence of B718P is provided in SEQ ID NO:504, with the cDNAsequence of the open reading frame including stop codon being providedin SEQ ID NO:505 and the cDNA sequence of the open reading frame withoutstop codon being provided in SEQ ID NO:506. The full-length amino acidsequence of B718P is provided is SEQ ID NO:507. SEQ ID NO:508 representsamino acids 1-158 of B718P, and SEQ ID NO:509 represents amino acids159-243 of B718P.

To determine mRNA expression levels of the isolated cDNA clones, cDNAclones from the breast subtraction described above were randomly pickedand colony PCR amplified. Their mRNA expression levels in breast tumor,normal breast and various other normal tissues were determined usingmicroarray technology (Synteni, Palo Alto, Calif.). Briefly, the PCRamplification products were arrayed onto slides in an array format, witheach product occupying a unique location in the array. mRNA wasextracted from the tissue sample to be tested, reverse transcribed, andfluorescent-labeled cDNA probes were generated. The microarrays wereprobed with the labeled cDNA probes, the slides scanned and fluorescenceintensity was measured. Data was analyzed using Synteni providedGEMTOOLS Software. Of the seventeen cDNA clones examined, those of SEQID NO:40, 46, 59 and 73 were found to be over-expressed in breast tumorand expressed at low levels in all normal tissues tested (breast, PBMC,colon, fetal tissue, salivary gland, bone marrow, lung, pancreas, largeintestine, spinal cord, adrenal gland, kidney, pancreas, liver, stomach,skeletal muscle, heart, small intestine, skin, brain and human mammaryepithelial cells). The clones of SEQ ID NO:41 and 48 were found to beover-expressed in breast tumor and expressed at low levels in all othertissues tested, with the exception of bone marrow. The clone of SEQ IDNO:42 was found to be over-expressed in breast tumor and expressed atlow levels in all other tissues tested except bone marrow and spinalcord. The clone of SEQ ID NO:43 was found to be over-expressed in breasttumor and expressed at low levels in all other tissues tested with theexception of spinal cord, heart and small intestine. The clone of SEQ IDNO:51 was found to be over-expressed in breast tumor and expressed atlow levels in all other tissues tested with the exception of largeintestine. The clone of SEQ ID NO:54 was found to be over-expressed inbreast tumor and expressed at low levels in all other tissues testedwith the exception of PBMC, stomach and small intestine. The clone ofSEQ ID NO:56 was found to be over-expressed in breast tumor andexpressed at low levels in all other tissues tested with the exceptionof large and small intestine, human mammary epithelia cells and SCIDmouse-passaged breast tumor. The clone of SEQ ID NO:60 was found to beover-expressed in breast tumor and expressed at low levels in all othertissues tested with the exception of spinal cord and heart. The clone ofSEQ ID NO:61 was found to be over-expressed in breast tumor andexpressed at low levels in all other tissues tested with the exceptionof small intestine. The clone of SEQ ID NO:72 was found to beover-expressed in breast tumor and expressed at low levels in all othertissues tested with the exception of colon and salivary gland.

The results of a Northern blot analysis of the clone SYN18C6 (SEQ IDNO:40) are shown in FIG. 1. A predicted protein sequence encoded bySYN18C6 is provided in SEQ ID NO:62.

Additional cDNA clones that are over-expressed in breast tumor tissuewere isolated from breast cDNA subtraction libraries as follows. Breastsubtraction libraries were prepared, as described above, by PCR-basedsubtraction employing pools of breast tumor cDNA as the tester and poolsof either normal breast cDNA or cDNA from other normal tissues as thedriver. cDNA clones from breast subtraction were randomly picked andcolony PCR amplified and their mRNA expression levels in breast tumor,normal breast and various other normal tissues were determined using themicroarray technology described above. Twenty-four distinct cDNA cloneswere found to be over-expressed in breast tumor and expressed at lowlevels in all normal tissues tested (breast, brain, liver, pancreas,lung, salivary gland, stomach, colon, kidney, bone marrow, skeletalmuscle, PBMC, heart, small intestine, adrenal gland, spinal cord, largeintestine and skin). The determined cDNA sequences for these clones areprovided in SEQ ID NO:63-87. Comparison of the sequences of SEQ IDNO:74-87 with those in the gene bank as described above, revealedhomology to previously identified human genes. No significant homologieswere found to the sequences of SEQ ID NO:63-73.

Three DNA isoforms for the clone B726P (partial sequence provided in SEQID NO:71) were isolated as follows. A radioactive probe was synthesizedfrom B726P by excising B726P DNA from a pT7Blue vector (Novagen) by aBamHI/XbaI restriction digest and using the resulting DNA as thetemplate in a single-stranded PCR in the presence of [α-32P]dCTP. Thesequence of the primer employed for this PCR is provided in SEQ IDNO:177. The resulting radioactive probe was used to probe a directionalcDNA library and a random-primed cDNA library made using RNA isolatedfrom breast tumors. Eighty-five clones were identified, excised,purified and sequenced. Of these 85 clones, three were found to eachcontain a significant open reading frame. The determined cDNA sequenceof the isoform B726P-20 is provided in SEQ ID NO:175, with thecorresponding predicted amino acid sequence being provided in SEQ IDNO:176. The determined cDNA sequence of the isoform B726P-74 is providedin SEQ ID NO:178, with the corresponding predicted amino acid sequencebeing provided in SEQ ID NO:179. The determined cDNA sequence of theisoform B726P-79 is provided in SEQ ID NO:180, with the correspondingpredicted amino acid sequence being provided in SEQ ID NO:181.

Efforts to obtain a full-length clone of B726P using standard techniquesled to the isolation of five additional clones that represent additional5′ sequence of B726P. These clones appear to be alternative splice formsof the same gene. The determined cDNA sequences of these clones areprovided in SEQ ID NO:464-468, with the predicted amino acid sequencesencoded by SEQ ID NO: 464-467 being provided in SEQ ID NO:470-473,respectively. Using standard computer techniques, a 3,681 bp consensusDNA sequence (SEQ ID NO:463) was created that contains two large openreading frames. The downstream ORF encodes the amino acid sequence ofSEQ ID NO:176. The predicted amino acid sequence encoded by the upstreamORF is provided in SEQ ID NO:469. Subsequent studies led to theisolation of an additional splice form of B726P that has 184 bp insertrelative to the other forms. This 184 bp insert causes a frameshift thatbrings the down stream and upstream ORFs together into a single ORF thatis 1002 aa in length. The determined cDNA sequence of this alternativesplice form is disclosed in SEQ ID NO:474, with the corresponding aminoacid sequence being provided in SEQ ID NO:475.

Comparison of the cDNA sequence of SEQ ID NO:63 (referred to as B723P)with the sequences in the GeneSeq™ DNA database showed matches to 5 DNAsequences (Accession nos. A26456, A37144, A26424, V84525 and T22133), 4of which appear to represent the full-length sequence of the gene. Threeof these sequences encode a 243 amino acid open reading frame (ORF),while one of the DNA sequences (Accession no. A37144) contains an extraC at position 35, resulting in a 278 amino acid ORF. The open readingframe, including stop codon, of the first variant of B723P (referred toas B723P-short) is provided in SEQ ID NO:510, with the open readingframe without stop codon being provided in SEQ ID NO:511. The openreading frame, including stop codon, of the second variant of B723P(referred to as B723P-long) is provided in SEQ ID NO:512, with the openreading frame without stop codon being provided in SEQ ID NO:513. Theamino acid sequences of B723P-short and B723P-long are provided in SEQID NO:514 and 515, respectively. Computer analysis of these sequencesdemonstrated the presence of putative transmembrane domains at aminoacids 233-252 of the B723P-long ORF and amino acids 198-217 of theB723P-short ORF. SEQ ID NO:516, 518 and 519 represent amino acids 1-197,198-243 and 218-243, respectively of B723P-short. SEQ ID NO:517represents amino acids 1-232 of B723P-long.

Further isolation of individual clones that are over-expressed in breasttumor tissue was conducted using cDNA subtraction library techniquesdescribed above. In particular, a cDNA subtraction library containingcDNA from breast tumors subtracted with five other normal human tissuecDNAs (brain, liver, PBMC, pancreas and normal breast) was utilized inthis screening. From the original subtraction, one hundred seventy sevenclones were selected to be further characterized by DNA sequencing andmicroarray analysis. Microarray analysis demonstrated that the sequencesin SEQ ID NO:182-251 and 479 were 2 or more fold over-expressed in humanbreast tumor tissues over normal human tissues. No significanthomologies were found for nineteen of these clones, including, SEQ IDNO:185, 186, 194, 199, 205, 208, 211, 214-216, 219, 222, 226, 232, 236,240, 241, 245, 246 and 479, with the exception of some previouslyidentified expressed sequence tags (ESTs). The remaining clones sharesome homology to previously identified genes, specifically SEQ IDNO:181-184, 187-193, 195-198, 200-204, 206, 207, 209, 210, 212, 213,217, 218, 220, 221, 223-225, 227-231, 233-235, 237-239, 242-244 and247-251.

One of the cDNA clones isolated by PCR subtraction as described above(SEQ ID NO:476; referred to as B720P) which was shown by microarray tobe over-expressed in breast tumor tissues, was found to be identical toa known keratin gene. The full-length cDNA sequence of the known keratingene is provided in SEQ ID NO:477, with the corresponding amino acidsequence being provided in SEQ ID NO:478. Primers were generated basedon the sequence of SEQ ID NO:477 and used to clone full-length cDNA frommRNA which was obtained from total RNA showing high expression of B720Pin real-time PCR analysis. Products were then cloned and sequenced. Thedetermined full-length cDNA sequence for B720P is provided in SEQ IDNO:484, with the corresponding amino acid sequence being provided in SEQID NO:485.

In further studies, a-truncated form of B720P (referred to as B720P-tr)was identified in breast carcinomas. This antigen was cloned from mRNAderived from total breast tumor RNA that showed high expression ofB720P-tr in real-time PCR analysis. mRNA was used to generate a pool ofcDNA which was then used as a template to amplify the cDNA correspondingto B720P-tr by PCR. The determined cDNA sequence for B720P-tr isprovided in SEQ ID NO:486. B720P-tr has an ORF of 708 base pairs whichencodes a 236 amino acid protein (SEQ ID NO:487). The size of thetranscript was confirmed by northern analysis.

Of the seventy clones showing over-expression in breast tumor tissues,fifteen demonstrated particularly good expression levels in breast tumorover normal human tissues. The following eleven clones did not show anysignificant homology to any known genes. Clone 19463.1 (SEQ ID NO:185)was over-expressed in the majority of breast tumors and also in the SCIDbreast tumors tested (refer to Example 2); additionally, over-expressionwas found in a majority of normal breast tissues. Clone 19483.1 (SEQ IDNO:216) was over-expressed in a few breast tumors, with noover-expression in any normal tissues tested. Clone 19470.1 (SEQ IDNO:219) was found to be slightly over-expressed in some breast tumors.Clone 19468.1 (SEQ ID NO:222) was found to be slightly over-expressed inthe majority of breast tumors tested. Clone 19505.1 (SEQ ID NO:226) wasfound to be slightly over-expressed in 50% of breast tumors, as well asin SCID tumor tissues, with some degree of over-expression in found innormal breast. Clone 1509.1 (SEQ ID NO:232) was found to beover-expressed in very few breast tumors, but with a certain degree ofover-expression in metastatic breast tumor tissues, as well as nosignificant over-expression found in normal tissues. Clone 19513.1 (SEQID NO:236) was shown to be slightly over-expressed in few breast tumors,with no significant over-expression levels found in normal tissues.Clone 19575.1 (SEQ ID NO:240) showed low level over-expression in somebreast tumors and also in normal breast. Clone 19560.1 (SEQ ID NO:241)was over-expressed in 50% of-breast tumors tested, as well as in somenormal breast tissues. Clone 19583.1 (SEQ ID NO:245) was slightlyover-expressed in some breast tumors, with very low levels ofover-expression found in normal tissues. Clone 19587.1 (SEQ ID NO:246)showed low level over-expression in some breast tumors and nosignificant over-expression in normal tissues.

Clone 19520.1 (SEQ ID NO:233), showing homology to clone 102D24 onchromosome 11q13.31, was found to be over-expressed in breast tumors andin SCID tumors. Clone 19517.1 (SEQ ID NO:237), showing homology to humanPAC 128M19 clone, was found to be slightly over-expressed in themajority of breast tumors tested. Clone 19392.2 (SEQ ID NO:247), showinghomology to human chromosome 17, was shown to be over-expressed in 50%of breast tumors tested. Clone 19399.2 (SEQ ID NO:250), showing homologyto human Xp22 BAC GSHB-184P14, was shown to be slightly over-expressedin a limited number of breast tumors tested.

In subsequent studies, 64 individual clones were isolated from asubtracted cDNA library containing cDNA from a pool of breast tumorssubtracted with cDNA from five normal tissues (brain, liver, PBMC,pancreas and normal breast). The subtracted cDNA library was prepared asdescribed above with the following modification. A combination of fivesix-base cutters (MluI, MscI, PvuII, SalI and StuI) was used to digestthe cDNA instead of RsaI. This resulted in an increase in the averageinsert size from 300 bp to 600 bp. The 64 isolated clones were colonyPCR amplified and their mRNA expression levels in breast tumor tissue,normal breast and various other normal tissues were examined bymicroarray technology as described above. The determined cDNA sequencesof 11 clones which were found to be over-expressed in breast tumortissue are provided in SEQ ID NO:405-415. Comparison of these sequencesto those in the public database, as outlined above, revealed homologiesbetween the sequences of SEQ ID NO:408, 411, 413 and 414 and previouslyisolated ESTs. The sequences of SEQ ID NO:405-4071 409, 410, 412 and 415were found to show some homology to previously identified sequences.

In further studies, a subtracted cDNA library was prepared from cDNAfrom metastatic breast tumors subtracted with a pool of cDNA from fivenormal tissues (breast, brain, lung, pancreas and PBMC) using thePCR-subtraction protocol of Clontech, described above. The determinedcDNA sequences of 90 clones isolated from this library are provided inSEQ ID NO:316-404. Comparison of these sequences with those in thepublic database, as described above, revealed no significant homologiesto the sequence of SEQ ID NO:366. The sequences of SEQ ID NO:321-325,343, 354, 368, 369, 377, 382, 385, 389, 395, 397 and 400 were found toshow some homology to previously isolated ESTs. The remaining sequenceswere found to show homology to previously identified gene sequences.

In yet further studies, a subtracted cDNA library (referred to as 2BT)was prepared from cDNA from breast tumors subtracted with a pool of cDNAfrom six normal tissues (liver, brain, stomach, small intestine, kidneyand heart) using the PCR-subtraction protocol of Clontech, describedabove. cDNA clones isolated from this subtraction were subjected to DNAmicroarray analysis as described above and the resulting data subjectedto four modified Gemtools analyses. The first analysis compared 28breast tumors with 28 non-breast normal tissues. A mean over-expressionof at least 2.1 fold was used as a selection cut-off. The secondanalysis compared 6 metastatic breast tumors with 29 non-breast normaltissues. A mean over-expression of at least 2.5 fold was used as acut-off. The third and fourth analyses compared 2 early SCIDmouse-passaged with 2 late SCID mouse-passaged tumors. A meanover-expression in the early or late passaged tumors of 2.0-fold orgreater was used as a cut-off. In addition, a visual analysis wasperformed on the microarray data for the 2BT clones. The determined cDNAsequences of 13 clones identified in the visual analysis are provided inSEQ ID NO:427-439. The determined cDNA sequences of 22 clones identifiedusing the modified Gemtools analysis are provided in SEQ ID NO:440-462,wherein SEQ ID NO:453 and 454 represent two partial, non-overlapping,sequences of the same clone.

Comparison of the clone sequences of SEQ ID NO:436 and 437 (referred toas 263G6 and 262B2) with those in the public databases, as describedabove, revealed no significant homologies to previously identifiedsequences. The sequences of SEQ ID NO:427, 429, 431, 435, 438, 441, 443,444, 445, 446, 450, 453 and 454 (referred to as 266B4, 266G3, 264B4,263G1, 262B6, 2BT2-34, 2BT1-77, 2BT1-62, 2BT1-60,61, 2BT1-59, 2BT1-52and 2BT1-40, respectively) showed some homology to previously isolatedexpressed sequences tags (ESTs). The sequences of SEQ ID NO:428, 430,432, 433, 434, 439, 440, 442, 447, 448, 449, 451, 452 and 455-462(referred to as clones 22892, 22890, 22883, 22882, 22880, 22869, 21374,21349, 21093, 21091, 21089, 21085, 21084, 21063, 21062, 21060, 21053,21050, 21036, 21037 and 21048, respectively), showed some homology togene sequences previously identified in humans.

EXAMPLE 2 Isolation and Characterization of Breast Tumor PolypeptidesObtained by PCR-Based Subtraction Using SCID-Passaged Tumor RNA

Human breast tumor antigens were obtained by PCR-based subtraction usingSCID mouse passaged breast tumor RNA as follows. Human breast tumor wasimplanted in SCID mice and harvested on the first or sixth serialpassage, as described in patent application Ser. No. 08/556,659 filedNov. 13, 1995, U.S. Pat. No. 5,986,170. Genes found to be differentiallyexpressed between early and late passage SCID tumor may be stagespecific and therefore useful in therapeutic and diagnosticapplications. Total RNA was prepared from snap frozen SCID passagedhuman breast tumor from both the first and sixth passage.

PCR-based subtraction was performed essentially as described above. Inthe first subtraction (referred to as T9), RNA from first passage tumorwas subtracted from sixth passage tumor RNA to identify more aggressive,later passage-specific antigens. Of the 64 clones isolated and sequencedfrom this subtraction, no significant homologies were found to 30 ofthese clones, hereinafter referred to as: 13053, 13057, 13059, 13065,13067, 13068, 13071-13073, 13075, 13078, 13079, 13081, 13082, 13092,13097, 13101, 13102, 13131, 13133, 13119, 13135, 13139, 13140,13146-13149, and 13151, with the exception of some previously identifiedexpressed sequence tags (ESTs). The determined cDNA sequences for theseclones are provided in SEQ ID NO:88-116, respectively. The isolated cDNAsequences of SEQ ID NO:117-140 showed homology to known genes.

In a second PCR-based subtraction, RNA from sixth passage tumor wassubtracted from first passage tumor RNA to identify antigensdown-regulated over multiple passages. Of the 36 clones isolated andsequenced, no significant homologies were found to nineteen of theseclones, hereinafter referred to as: 14376, 14377, 14383, 14384, 14387,14392, 14394, 14398, 14401, 14402, 14405,1 4409, 14412, 14414-14416,14419, 14426, and 14427, with the exception of some previouslyidentified expressed sequence tags (ESTs). The determined cDNA sequencesfor these clones are provided in SEQ ID NO:141-159, respectively. Theisolated cDNA sequences of SEQ ID NO: 160-174 were found to showhomology to previously known genes.

Further analysis of human breast tumor antigens through PCR-basedsubtraction using first and sixth passage SCID tumor RNA was performed.Sixty three clones were found to be differentially expressed by a two ormore fold margin, as determined by microarray analysis, i.e., higherexpression in early passage tumor over late passage tumor, or viceversa. Seventeen of these clones showed no significant homology to anyknown genes, although some degree of homology with previously identifiedexpressed sequence tags (ESTs) was found, hereinafter referred to as20266, 20270, 20274, 20276, 20277, 20280, 20281, 20294, 20303, 20310,20336, 20341, 20941, 20954, 20961, 20965 and 20975 (SEQ ID NO:252-268,respectively). The remaining clones were found to share some degree ofhomology to known genes, which are identified in the Brief Descriptionof the Drawings and Sequence Identifiers section above, hereinafterreferred to as 20261, 20262, 20265, 20267, 20268, 20271, 20272, 20273,20278, 20279, 20293, 20300, 20305, 20306, 20307, 20313, 20317, 20318,20320, 20321, 20322, 20326, 20333, 20335, 20337, 20338, 20340, 20938,20939, 20940, 20942, 20943, 20944, 20946, 20947, 20948, 20949, 20950,20951, 20952, 20957, 20959, 20966, 20976, 20977 and 20978. Thedetermined cDNA sequences for these clones are provided in SEQ IDNO:269-314, respectively.

The clones 20310, 20281, 20262, 20280, 20303, 20336, 20270, 20341, 20326and 20977 (also referred to as B820P, B821P, B822P, B823P, B824P, B825P,B826P, B827P, B828P and B829P, respectively) were selected for furtheranalysis based on the results obtained with microarray analysis.Specifically, microarray data analysis indicated at least two- tothree-fold overexpression of these clones in breast tumor RNA comparedto normal tissues tested. Subsequent studies led to the determination ofthe complete insert sequence for the clones B820P, B821P, B822P, B823P,B824P, B825P, B826P, B827P, B828P and B829P. These extended cDNAsequences are provided in SEQ ID NO:416-426, respectively.

EXAMPLE 3 Synthesis of Polypeptides

Polypeptides may be synthesized on an Perkin Elmer/Applied BiosystemsDivision 430A peptide synthesizer using FMOC chemistry with HPTU(O-Benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate)activation. A Gly-Cys-Gly sequence may be attached to the amino terminusof the peptide to provide a method of conjugation, binding to animmobilized surface, or labeling of the peptide. Cleavage of thepeptides from the solid support may be carried out using the followingcleavage mixture: trifluoroaceticacid:ethanedithiol:thioanisole:water:phenol (40:1:2:2:3). After cleavingfor 2 hours, the peptides may be precipitated in coldmethyl-t-butyl-ether. The peptide pellets may then be dissolved in watercontaining 0.1% trifluoroacetic acid (TFA) and lyophilized prior topurification by C18 reverse phase HPLC. A gradient of 0%-60%acetonitrile (containing 0.1% TFA) in water (containing 0.1% TFA) may beused to elute the peptides. Following lyophilization of the purefractions, the peptides may be characterized using electrospray or othertypes of mass spectrometry and by amino acid analysis.

EXAMPLE 4 elicitation of Breast Antigen-Specific CTL Responses in HumanBlood

This Example illustrates the ability of the breast-specific antigenB726P to elicit a cytotoxic T lymphocyte (CTL) response in peripheralblood lymphocytes from normal humans.

Autologous dendritic cells (DC) were differentiated from monocytecultures derived from PBMC of a normal donor by growth for five days inRPMI medium containing 10% human serum, 30 ng/ml GM-CSF and 30 ng/mlIL-4. Following five days of culture, DC were infected overnight withadenovirus expressing recombinant B726P (downstream ORF; SEQ ID NO:176)at an M.O.I. of 2.5 and matured for 8 hours by the addition of 2micrograms/ml CD40 ligand. CD8 positive cells were enriched for by thedepletion of CD4 and CD14-positive cells. Priming cultures wereinitiated in individual wells of several 96-well plates with thecytokines IL-6 and IL-12. These cultures were restimulated in thepresence of IL-2 using autologous fibroblasts treated with IFN-gamma andtransduced with B726P and CD80. Following three stimulation cycles, thepresence of B726P-specific CTL activity was assessed in IFN-gammaElispot assays (Lalvani et al., J. Exp. Med. 186:859-865, 1997) usingIFN-gamma treated autologous fibroblasts transduced to express eitherB726P or an irrelevant, control, antigen as antigen presenting cells(APC). Of approximately 96 lines, one line (referred to as 6-2B) wasidentified that appeared to specifically recognize B726P-transduced APCbut not control antigen-transduced APC. This microculture was clonedusing standard protocols. B726P-specific CTL were identified by Elispotanalysis and expanded for further analysis. These CTL clones weredemonstrated to recognize B726P-expressing fibroblasts, but not thecontrol antigen MART-1, using chromium-51 release assays. Furthermore,using a panel of allogeneic fibroblasts transduced with B726P inantibody blocking assays, the HLA restriction element for theseB726P-specific CTL was identified as HLA-B*1501.

In order to define more accurately the location of the epitoperecognized by the B726P-specific CTL clones, a deletion constructcomprising only the N-terminal half (a.a. 1-129) of B726P (referred toas B726Pdelta3′) was constructed in the pBIB retroviral expressionplasmid. This plasmid, as well as other plasmids containing B726P, weretransfected into COS-7 cells either alone or in combination with aplasmid expressing HLA-B*1501. Aproximately 48 hours after transfection,a B726P-specific CTL clone (1-9B) was added at approximately 10⁴ cellsper well. The cells were harvested the next day and the amount ofIFN-gamma released was measured by ELISA. The CTL responded abovebackground (EGFP) to COS-7 cells that had been transfected with bothB726P and HLA-B*1501. There was no response above background to COS-7cells that had been transfected with either B726P or HLA-B*1501 alone.Importantly, a higher response was seen with COS-7 cells that had beentransfected with both HLA-B*1501 and B726Pdelta3′. This result indicatedthat the epitope was likely to be located in the N-terminal region (a.a.1-129) of B726P. This region was examined and amino acid sequences thatcorresponded to the HLA-B*1501 peptide binding motif (J.Immunol.1999,162:7277-84) were identified and synthesized. Thesepeptides were pulsed at 10 ug/ml onto autologous B-LCL overnight. Thenext day, the cells were washed and the ability of the cells tostimulate the B726P-specific CTL clone 1-9B was assayed in a IFN-gammaELISPOT assay. Of the eleven peptides tested, only one peptide, havingthe amino acid sequence SLTKRASQY (a.a. 76-84 of B726P; SEQ ID NO: 488)was recognized by the CTL clone. This result identifies this peptide asbeing a naturally-processed epitope recognized by this B726P-specificCTL clone.

In further studies, a panel of breast tumor cell lines obtained from theAmerican Type Culture Collection (Manassas, Va.), was analyzed usingreal time PCR to determine their B726P message level. The cell line thatexpressed the highest level of B726P (referred to as HTB21) and a linethat expressed no B726P (referred to as HTB132) were transduced withHLA-B*1501. These cell lines were grown up and analyzed using FACS todetermine their B1501 expression. The line HTB 21 was found toendogenously express B1501. To determine if clone 1-9A would recognizethe tumor cell line HTB2 1, an IFN-gamma ELISPOT assay was performedusing 20,000 T cells, low dose IL-2 (5 ug/ml), and 20,000 of thefollowing targets: autologous B726P or Mart-1 fibroblasts, untransducedor B1501-transduced HTB21; or untransduced or B1501-transduced HTB132.These were incubated overnight and the assay was developed the next day.The results of this assay are shown in FIG. 2. These studies demonstratethat B726P-specific CTL can recognize and lyse breast tumor cellsexpressing B726P.

EXAMPLE 5 Identification of Immunogenic CD4 T Cell Epitopes in BreastAntigens

Immunogenic CD4 T cell epitopes derived from the breast antigen B726Pwere identified as follows.

A total of thirty-five 20-mer peptides overlapping by 12 amino acids andderived from the downstream ORF of B726P (corresponding to amino acids1-317 of SEQ ID NO:176) were generated by standard procedure. Dendriticcells (DC) were derived from PBMC of a normal male donor using GMCSF andIL-4 by standard protocol. Purified CD4 T cells were generated from thesame donor as the DC using MACS beads and negative selection of PBMCs.DC were pulsed overnight with pools of the 20-mer peptides, with eachpeptide at an individual concentration of 0.5 micrograms/mL. Pulsed DCwere washed and plated at 10,000 cells/well of 96-well U bottom plates,and purified CD4 T cells were added at 100,000 cells/well. Cultures weresupplemented with 10 ng/mL IL-6 and 5 ng/mL IL-12 and incubated at 37°C. Cultures were restimulated as above on a weekly basis using DC madeand pulsed as above as the antigen presenting cell, supplemented with 10u/mL IL-2 and 5 ng/mL IL-7. Following three in vitro stimulation cycles(the initial priming+two restimulations), cell lines (each correspondingto one well) were tested for specific proliferation and cytokineproduction in response to the stimulating pool versus an irrelevant poolof peptides derived from unrelated antigens. A number of individual CD4T cell lines (36/672 by IFN-gamma and 64/672 by proliferation)demonstrated significant cytokine release (IFN-gamma) and proliferationin response to the B726P peptide pools but not to the control peptidepool. Twenty-five of these T cell lines were restimulated on theappropriate pool of B726P peptides and reassayed on autologous DC pulsedwith either the individual peptides or recombinant B726P protein made inE. coli. Approximately 14 immunogenic peptides were recognized by the Tcells from the entire set of peptide antigens tested. The amino acidsequences of these 14 peptides are provided in SEQ ID NO:534-547, withthe corresponding DNA sequences being provided in SEQ ID NO:520-533,respectively. In some cases the peptide reactivity of the T cell linecould be mapped to a single peptide but some could be mapped to morethan one peptide in each pool. Thirteen of the fifteen T cell linesrecognized the recombinant B726P protein. These results demonstrate that13 of the 14 peptide sequences (SEQ ID NO:534-542 and 544-547) may benaturally processed CD4 epitopes of the B726P protein.

EXAMPLE 6 Preparation and Characterization of Antibodies Against BreastTumor Antigen B726P

Polyclonal antibodies against both the downstream (SEQ ID NO:176) andupstream (SEQ ID NO:469) ORF of the breast tumor antigen B726P wereprepared as follows.

The downstream or upstream ORF of B726P expressed in an E. colirecombinant expression system was grown overnight in LB broth with theappropriate antibiotics at 37° C. in a shaking incubator. The nextmorning, 10 ml of the overnight culture was added to 500 ml to 2× YTplus appropriate antibiotics in a 2L-baffled Erlenmeyer flask. When theOptical Density (at 560 nm) of the culture reached 0.4-0.6, the cellswere induced with IPTG (1 mM). Four hours after induction with IPTG, thecells were harvested by centrifugation. The cells were then washed withphosphate buffered saline and centrifuged again. The supernatant wasdiscarded and the cells were either frozen for future use or immediatelyprocessed. Twenty ml of lysis buffer was added to the cell pellets andvortexed. To break open the E. coli cells, this mixture was then runthrough the French Press at a pressure of 16,000 psi. The cells werethen centrifuged again and the supernatant and pellet were checked bySDS-PAGE for the partitioning of the recombinant protein. For proteinsthat localized to the cell pellet, the pellet was resuspended in 10 mMTris pH 8.0, 1% CHAPS and the inclusion body pellet was washed andcentrifuged again. This procedure was repeated twice more. The washedinclusion body pellet was solubilized with either 8 M urea or 6 Mguanidine HCl containing 10 mM Tris pH 8.0 plus 10 mM imidazole. Thesolubilized protein was added to 5 ml of nickel-chelate resin (Qiagen)and incubated for 45 min to 1 hour at room temperature with continuousagitation. After incubation, the resin and protein mixture were pouredthrough a disposable column and the flow through was collected. Thecolumn was then washed with 10-20 column volumes of the solubilizationbuffer. The antigen was then eluted from the column using 8M urea, 10 mMTris pH 8.0 and 300 mM imidazole and collected in 3 ml fractions. ASDS-PAGE gel was run to determine which fractions to pool for furtherpurification.

As a final purification step, a strong anion exchange resin, such asHiPrepQ (Biorad), was equilibrated with the appropriate buffer and thepooled fractions from above were loaded onto the column. Antigen waseluted off the column with a increasing salt gradient. Fractions werecollected as the column was run and another SDS-PAGE gel was run todetermine which fractions from the column to pool. The pooled fractionswere dialyzed against 10 mM Tris pH 8.0. The protein was then vialedafter filtration through a 0.22 micron filter and the antigens werefrozen until needed for immunization.

Four hundred micrograms of the B726P antigen was combined with 100micrograms of muramyldipeptide (MDP). Every four weeks rabbits wereboosted with 100 micrograms mixed with an equal volume of IncompleteFreund's Adjuvant (IFA). Seven days following each boost, the animal wasbled. Sera was generated by incubating the blood at 4° C. for 12-24hours followed by centrifugation.

Ninety-six well plates were coated with B726P antigen by incubating with50 microliters (typically 1 microgram) of recombinant protein at 4° C.for 20 hours. 250 Microliters of BSA blocking buffer was added to thewells and incubated at room temperature for 2 hours. Plates were washed6 times with PBS/0.01% Tween. Rabbit sera was diluted in PBS. Fiftymicroliters of diluted sera was added to each well and incubated at roomtemperature for 30 min. Plates were washed as described above before 50microliters of goat anti-rabbit horse radish peroxidase (HRP) at a1:10000 dilution was added and incubated at room temperature for 30 min.Plates were again washed as described above and 100 microliters of TMBmicrowell peroxidase substrate was added to each well. Following a 15min incubation in the dark at room temperature, the colorimetricreaction was stopped with 100 microliters of 1N H₂SO₄ and readimmediately at 450 nm. All the polyclonal antibodies showedimmunoreactivity to the appropriate B726P antigen.

B) Preparation of Polyclonal Antibodies Against B709P and B720P

The breast antigens B709P (SEQ ID NO: 62) and B720P (SEQ ID NO: 485)expressed in an E. coli recombinant expression system were grownovernight in LB Broth with the appropriate antibiotics at 37° C. in ashaking incubator. Ten ml of the overnight culture was added to 500 mlof 2×YT plus appropriate antibiotics in a 2L-baffled Erlenmeyer flask.When the optical density (at 560 nanometers) of the culture reached0.4-0.6, the cells were induced with IPTG (1 mM). Four hours afterinduction with IPTG, the cells were harvested by centrifugation. Thecells were washed with phosphate buffered saline and centrifuged again.The supernatant was discarded and the cells were either frozen forfuture use or immediately processed. Twenty milliliters of lysis bufferwas added to the cell pellets and vortexed. To break open the E. colicells, the mixture was run through a French Press at a pressure of16,000 psi. The cells were centrifuged again and the supernatant andpellet were checked by SDS-PAGE for the partitioning of the recombinantprotein. For proteins that localized to the cell pellet, the pellet wasresuspended in 10 mM Tris pH 8.0, 1% CHAPS and the inclusion body pelletwas washed and centrifuged again. This procedure was repeated twicemore. The washed inclusion body pellet was solubilized with either 8 Murea or 6 M guanidine HCl containing 10 mM Tris pH 8.0 plus 10 mMimidazole. The solubilized protein was added to 5 ml of nickel-chelateresin (Qiagen) and incubated for 45 min to 1 hour at room temperature(RT) with continuous agitation. After incubation, the resin and proteinmixture were poured through a disposable column and the flow through wascollected. The column was then washed with 10-20 column volumes of thesolubilization buffer. The antigen was then eluted from the column using8M urea, 10 mM Tris pH 8.0 and 300 mM imidazole and collected in 3 mlfractions. A SDS-PAGE gel was run to determine which fractions to poolfor further purification. As a final purification step, a strong anionexchange resin such as Hi-Prep Q (Biorad) was equilibrated with theappropriate buffer and the pooled fractions from above were loaded ontothe column. Each antigen was eluted off of the column with an increasingsalt gradient. Fractions were collected as the column was run andanother SDS-PAGE gel was run to determine which fractions from thecolumn to pool. The pooled fractions were dialyzed against 10 mM Tris pH8.0. The proteins were then vialed after filtration through a0.22-micron filter and frozen until needed for immunization.

Four hundred micrograms of antigen was combined with 100 micrograms ofmuramyldipeptide (MDP). An equal volume of Incomplete Freund's Adjuvant(IFA) was added and mixed, and the mixture was injected into a rabbit.The rabbit was boosted with 100 micrograms of antigen mixed with anequal volume of IFA every four weeks. The animal was bled seven daysfollowing each boost. Sera was generated by incubating the blood at 4°C. for 12-24 hours followed by centrifugation.

The reactivity of the polyclonal antibodies to recombinant antigen(B709P or B720P) was determined by ELISA as follows. Ninety-six wellplates were coated with antigen by incubating with 50 microliters(typically 1 microgram) at 4° C. for 20 hrs. 250 microliters of BSAblocking buffer was added to the wells and incubated at RT for 2 hrs.Plates were washed 6 times with PBS/0.01% Tween. Rabbit sera werediluted in PBS. Fifty microliters of diluted sera was added to each welland incubated at RT for 30 min. Plates were washed as described abovebefore 50 microliters of goat anti-rabbit horse radish peroxidase (HRP)at a 1:10000 dilution was added and incubated at RT for 30 min. Plateswere washed as described above and 100 microliters of TMB MicrowellPeroxidase Substrate was added to each well. Following a 15-minuteincubation in the dark at RT, the colorimetric reaction was stopped with100 microliters of 1N H₂SO₄ and read immediately at 450 nm. Thepolyclonal antibodies showed immunoreactivity to the appropriateantigen.

EXAMPLE 7 Protein Expression of Breast Tumor Antigens

The downstream ORF of B726P (SEQ ID NO:176), together with a C-terminal6×His Tag, was expressed in insect cells using the baculovirusexpression system as follows.

The cDNA for the full-length downstream ORF of B726P was PCR amplifiedusing the primers of SEQ ID NO:480 and 481. The PCR product with theexpected size was recovered from agarose gel, restriction digested withEcoRI and Hind II, and ligated into the transfer plasmid pFastBac1,which was digested with the same restriction enzymes. The sequence ofthe insert was confirmed by DNA sequencing. The recombinant transferplasmid pFBB726P was used to make recombinant bacmid DNA and virus usingthe Bac-To-Bac Baculovirus expression system (BRL Life Technologies,Gaithersburg, Md.). High Five cells were infected with the recombinantvirus BVB726P to produce protein. The cDNA and amino acid sequences ofthe expressed B726P recombinant protein are provided in SEQ ID NO:482and 483, respectively.

EXAMPLE 8 Generation of Constructs for Protein Expression of B726P in E.Coli

Three different open reading frames (ORFs) of B726P were subcloned intopPDM, a modified pET28 vector for exression in E. coli.

Construct for the expression of B726P Upstream ORF in E. coli (cDNA: SEQID NO:549; amino acid: SEQ ID NO:552):

The partial B726P upstream ORF (A) from clone 23113 was PCR amplifiedwith the following primers: PDM-416 (SEQ ID NO:554) 5′gtcggctccatgagtcccgcaaaag 3′ Tm 63° C. PDM-431 (SEQ ID NO:555) 5′cgagaattcaatacttaagaagaccatctttaccag 3′ Tm 61° C.

The amplification conditions were as follows 10 μl 10×Pfu buffer, 1 μl10 μM dNTPs, 2 μl 10 μM each oligo, 83 μl sterile water, 1.5 μl Pfu DNApolymerase (Stratagene, La Jolla, Calif.), 1 μl PCR 23113. The reactionwas first denatured for 2 minutes at 96° C., followed by 40 cylces of96° C for 20 seconds, 62° C. for 15 seconds, and extension at 72° C. for2 minutes. This was followed by a final extension of 72° C. for 4minutes.

The second partial B726P upstream ORF (B) from clone 19310 was PCRamplified with the following primers: PDM-432 (SEQ ID NO:556) 5′cataagcttaaggctaactgcggaatgaaag 3′ Tm 63° C. PDM-427 (SEQ ID NO:557) 5′cccgcagaattcaacatgcaattttcatgtaagag 3′ Tm 62° C.

The amplification and cycling conditions were as described above. Thefirst PCR product was digested with EcoRI and cloned into pPDM His (amodified pET28 vector) that had been digested with EcoRI and Eco72I. Thesecond PCR product was digested with BfrI and EcoRI and cloned into theresulting construct: pPDM B726P UP-A-5 at the EcoRI and BfrI sites. Theconstruct (pPDM B726P Up-4) was confirmed to be correct through sequenceanalysis and transformed into BL21 (DE3) pLys S and BL21 CodonPlus RIL(DE3) cells. Protein expression was confirmed by Coomassie stainedSDS-PAGE and N-terminal protein sequence analysis.

Construct for B726P D-ORF expression in E. coli (cDNA: SEQ ID NO:550;amino acid: SEQ ID NO:551):

The B726P D-ORF was PCR amplified with the following primers: PDM-290(SEQ ID NO:558) 5′ ctaaatgccggcacaagagctctgc 3′ Tm 61° C. PDM-291 (SEQID NO:559) 5′ cgcgcagaattctattatataacttctgtttctgc 3′ Tm 61° C.

The reaction conditions were as described. The cycling conditions werealtered slightly in that the annealing temperature was lowered to 61° C.from 62° C. and was held for 15 seconds. The extension time was alsoincreased to 2 minutes and 15 seconds. The PCR product was digested withNaeI and EcoRI and cloned into pPDM His which has been digested withEco72I and EcoRI. Construct was confirmed by sequencing and thentransformed into BL21 (DE3) pLys S cells (Novagen, Madison, Wis.).Protein expression was confirmed by Coomassie stained SDS-PAGE andN-terminal protein sequence analysis.

Construct for B726P Combined ORF expression in E. coli (cDNA: SEQ IDNO:548; amino acid: SEQ ID NO:553):

The B726P C-1 coding region was PCR amplified including the 183 bpinsert, with the following primers: PDM-750 (SEQ ID NO:560) 5′ggggaattgtgagcggataacaattc 3′ Tm 58° C. PDM-752 (SEQ ID NO:561) 5′cgtagaattcaacctgatttaaattactttctacac 3′ Tm 59° C.

The B726P Downstream ORF was PCR amplified with the following primers:PDM-753 (SEQ ID NO:562) 5′ gaaagtaatttaaatcaggtttctcacactc 3′ Tm 59° C.PDM-751 (SEQ ID NO:563) 5′ gaggccccaaggggttatgctag 3′ Tm 61° C.

The reaction conditions for these PCR reactions were the same asdescribed above. The cycling conditions were as follows: 1^(st) PCR: Thereaction was first denatured for 2 minutes at 96° C., followed by 40cylces of 96° C. for 20 seconds, 58° C. for 15 seconds, and extension at72° C. for 4 minutes. This was followed by a final extension of 72° C.for 4 minutes; 2^(nd) PCR. The reaction was first denatured for 2minutes at 96° C., followed by 40 cylces of 96° C. for 20 seconds, 59°C. for 15 seconds, and extension at 72° C. for 2 minutes. This wasfollowed by a final extension of 72° C. for 4 minutes. The first PCRproduct was digested with EcoRI and cloned into pPDM His (a modifiedpET28 vector) at the Eco 72I and EcoRI sites. The construct wasconfirmed to be correct through sequence analysis. The second PCRproduct was digested with EcoRI and cloned into pPDM His at the samesites. The resulting constructs pPDM B726P UA-8 and pPDM B726P DA-7respectively were digested with SwaI and EcoRI. The pPDM B726P UA-8construct was used as vector and the insert from the pPDM B726P DA-7 wascloned into this construct successfully. The construct was confirmed tobe correct through sequence analysis and then transformed into BLR (DE3)pLys S and HMS 174 (DE3) pLys S cells (Novagen, Madison, Wis.). Proteinexpression was confirmed by Coomassie stained SDS-PAGE and N-terminalprotein sequence analysis.

EXAMPLE 9 Additional Sequence Identified for Breast Tumor Antigen B726Pby Bioinformatic Analysis

The combined ORF of the breast tumor antigen, B726P (amino acid sequenceset forth in SEQ ID NO:475), was used to search public databases. Asequence essentially identical to the combined ORF with additionalN-terminal sequence was identified in the GenBank nonredundant proteindatabase and the cDNA and predicted amino acid sequences are set forthin SEQ ID NO:564 and 565, respectively. The gene is also referred to asNY-BR-1 and was described in described in Cancer Research61(5):2055-2061, Mar. 1, 2001.

EXAMPLE 10 Analysis of B726P Expression Using Immunohistochemistry

Affinity purified polyclonal antibodies anti-B726Pup (generated againstthe B726P upstream ORF protein) and anti-B726Pdown (generated againstthe B726P downstream ORF) were used to assess B726P protein expressionin breast cancer and in a variety of normal tissue sections.

In order to determine which tissues express the breast cancer antigenprotein B726P immunohistochemistry (IHC) analysis was performed on adiverse range of tissue sections. Tissue samples were fixed in formalinsolution for 12-24 hrs and embedded in paraffin before being sliced into8 micron sections. Steam heat induced epitope retrieval (SHIER) in 0.1 Msodium citrate buffer (pH 6.0) was used for optimal staining conditions.Sections were incubated with 10% serum/PBS for 5 minutes. Primaryantibody (either rabbit affinity purified anti-B726Pdown oranti-B726Pup) was added to each section for 25 minutes followed by 25minute incubation with anti-rabbit biotinylated antibody. Endogenousperoxidase activity was blocked by three 1.5 minute incubations withhydrogen peroxidase. The avidin biotin complex/horse radish peroxidase(ABC/HRP) system was used along with DAB chromogen to visualize antigenexpression. Slides were counterstainied with hematoxylin to visualizecell nuclei. Anti-B726Pup and anti-B726Pdown immunoreactivity wasobserved in about 30-40% of breast cancer samples analyzed but notobserved in a majority of various normal tissues. Anti-B726Pdown andanti-b726Pup also stained roughly the same breast cancer samples. Thus,these data confirm earlier microarray analysis (see Example 1) showingthat B726P is overexpressed in breast tumor tissue as compared to normaltissue. Therefore, this antigen may be used in diagnostic andimmunotherapeutic applications for breast cancer.

EXAMPLE 11 Generation of Monoclonal Antibodies to B726P Downstream andUpstream ORFs

Production and purification of protein used for antibody generation.B726 upstream ORF and B726 downstream ORF proteins were expressed in anE. coli recombinant expression system (see Example 8). Cells were grownovernight in LB Broth with the appropriate antibiotics at 37° C. in ashaking incubator. The next morning, 10 ml of the overnight culture wasadded to 500 ml of 2×YT plus appropriate antibiotics in a 2L-baffledErlenmeyer flask. When the optical density (at 560 nanometers) of theculture reached 0.4-0.6 the cells were induced with IPTG (1 mM). Fourhours after induction with IPTG the cells were harvested bycentrifugation. The cells were then washed with phosphate bufferedsaline and centrifuged again. The supernatant was discarded and thecells were either frozen for future use or immediately processed. Twentymilliliters of lysis buffer was added to the cell pellets and vortexed.To break open the E. coli cells, this mixture was then run through theFrench Press at a pressure of 16,000 psi. The cells were thencentrifuged again and the supernatant and pellet were checked bySDS-PAGE for the partitioning of the recombinant protein. For proteinsthat localized to the cell pellet, the pellet was resuspended in 10 mMTris pH 8.0, 1% CHAPS and the inclusion body pellet was washed andcentrifuged again. This procedure was repeated twice more. The washedinclusion body pellet was solubilized with either 8 M urea or 6 Mguanidine HCl containing 10 mM Tris pH 8.0 plus 10 mM imidazole.

The solubilized protein was added to 5 ml of nickel-chelate resin(Qiagen, Valencia, Calif.) and incubated for 45 min to 1 hour at roomtemperature with continuous agitation. After incubation, the resin andprotein mixture were poured through a disposable column and the flowthrough was collected. The column was then washed with 10-20 columnvolumes of the solubilization buffer. The antigen was then eluted fromthe column using 8M urea, 10 mM Tris pH 8.0 and 300 mM imidazole andcollected in 3 ml fractions. A SDS-PAGE gel was run to determine whichfractions to pool for further purification. As a final purificationstep, a strong anion exchange resin such as Hi-Prep Q (Biorad) wasequilibrated with the appropriate buffer and the pooled fractions fromabove were loaded onto the column. Each antigen was eluted off of thecolumn with an increasing salt gradient. Fractions were collected as thecolumn was run and another SDS-PAGE gel was run to determine whichfractions from the column to pool. The pooled fractions were dialyzedagainst 10 mM Tris pH 8.0. This material was then submitted to QualityControl for final release. The release criteria were purity asdetermined by SDS-PAGE or HPLC, concentration as determined by Lowryassay or Amino Acid Analysis, identity as determined by amino terminalprotein sequence, and endotoxin level as determined by the Limulus (LAL)assay. The proteins were then vialed after filtration through a0.22-micron filter and the antigens were frozen until needed forimmunization.

To generate anti-B726P mouse monoclonal antibodies, mice were immunizedIP with 50 micrograms of recombinant B726P upstream ORF and B726Pdownstream ORF proteins that had been mixed to form an emulsion with anequal volume of Complete Freund's Adjuvant (CFA). Every three weeksanimals were injected IP with 50 micrograms of recombinant B726Pupstream ORF and B726P downstream ORF that had been mixed with an equalvolume of IFA to form an emulsion. After the fourth injection, spleenswere isolated and standard hybridoma fusion procedures were used togenerate anti-B726P mouse monoclonal antibody hybridomas. Anti-B726Pmonoclonal antibodies were screened using the ELISA analysis using thebacterially expressed recombinant B726P upstream ORF and B726Pdownstream ORF proteins.

A list of the mouse anti-B726P monoclonal antibodies that weregenerated, as well as their anti-B726P reactivity in an ELISA assay andWestern blot are shown in Table 2. The hybridomas were then subclonedand the subclones further tested for reactivity with B726P upstream ORFand B726P downstream ORF proteins. Several monoclonal antibodies showedparticularly favorable reactivity: 220A2-21, 220A19-25, 220A94-29,220A151-33.

For Western blot analysis, recombinant B726P upstream ORF and B726Pdownstream ORF protein was diluted with SDS-PAGE loading buffercontaining beta-mercaptoethanol, then boiled for 10 minutes prior toloading the SDS-PAGE gel. Protein was transferred to nitrocellulose andprobed with each of the anti-B726P hybridoma supernatants. Protein A-HRPwas used to visualize the anti-B726P reactive bands by incubation in ECLsubstrate. TABLE 2 B726PUP AND B726PDOWN MONOCLONAL ANTIBODY REACTIVITYAnti- B726P ELISA Western Blots mAbs B726PDown B726PUp L523S B726PdownB726Pup 220A2 +++ + − +++ ++ 220A10 − − − N/A N/A 220A14 +++ +++ +++ +++++ 220A19 ++ − − ++ + 220A43 +++ + − +++ ++ 220A86 +++ + − +++ ++ 220A94+++ − − ++ +/− 220A123 ++ − − + − 220A139 +/− − − + − 220A140 − − − N/AN/A 220A141 − − − N/A N/A 220A143 − − − N/A N/A 220A151 ++ − − ++ −220A176 +/− − − + −

EXAMPLE 12 Identification of Additional Sequences for B726P

Additional 5′ sequence was obtained for B726P—this sequence was obtainedby PCR from 1st strand cDNA prepared from three separate mRNA sources(metastatic breast tumor, breast tumor, normal testis). Disclosed hereinare clones that were isolated, each with differences from the expectedpublished sequence of NY-BR-1.

A 1300 bp fragment of B726P otherwise known as NY-BR-1 was PCR amplifiedfrom 1st strand cDNA and cloned into pPDM, a modified pET28 vector asfollows:

The B726P XB coding region (NY-BR-1) was PCR amplified with thefollowing primers PDM-784 5′ cacacaaagaggaagaagaccatc 3′ Tm 56° C.PDM-814 5′ gattcttttgtaggacatgcaatcatc 3′ Tm 55° C.

The following PCR conditions were used: 10 μl 10×Herculase buffer, 1 μl10 mM dNTPs, 2 μl 10 .μM each oligo, 83 μl sterile water, 1.5 μlHerculase DNA polymerase (Stratagene, La Jolla, Calif.), 50 ng DNA. Thethermalcycling conditions were as follows: 98° C. 3 minutes 98° C. 40seconds, 51° C. 15 seconds, 72° C. 4 minutes, ×10 cycles 98° C. 40seconds, 51° C. 15 seconds, 72° C. 5 minutes, ×10 cycles 98° C. 40seconds, 51° C. 15 seconds, 72° C. 6 minutes, ×10 cycles 98° C. 40seconds, 51° C. 15 seconds, 72° C. 7 minutes, ×10 cycles 72° C. 10minutes

The PCR product was ligated into the pPDM vector (a modified pET28) thathad been digested with Eco72I and de-phosphorylated. PCR amplificationof this gene proved very difficult and required the use of a polymeraselacking proofreading capabilities. However, use of such an enzyme, inthis case, Herculase from Stratagene (La Jolla, Calif.), led to what islikely PCR errors in the resulting clones. The cDNA sequence of three ofthe isolated clones containing mutations are disclosed in SEQ IDNO:567-569 with the corresponding amino acid sequences disclosed in SEQID NO:572, 571, and 570, respectively.

The resulting construct, pPDM B726P XB (clone 83686), was then digestedwith BglII and the insert which dropped out from the 5′ vector BglIIsite and the internal BglII site at amino acids 390-391 was cloned intothe pPDM B726P C-ORF (SEQ ID NO:548) that had been digested with BglIIand was de-phosphorylated. This construct, pPDM B726P XC, was then DNAsequenced and showed two nucleotide changes, which result in two aminoacid changes. The cDNA of the full-length clone containing these 2mutations is disclosed in SEQ ID NO:566 with the corresponding aminoacid sequence in SEQ ID NO:573. The full-length expected, publishedNY-BR-1 is disclosed in SEQ ID NO:564 (cDNA); amino acid SEQ ID NO:565.

EXAMPLE 13 Isolation of Additional 3′ Sequence and Real-Time PCRAnalysis of B726P Homolog NY-BR1.1

A sequence homolog to the breast candidate B726P, called NY-BR-1.1, wasidentified and published in Cancer Research 61(5):2055-2061; Mar. 1,2001. The NY-BR-1.1 gene, thought to be located on chomosome 9 based on100% sequence identity to genomic sequence from chromosome 9, was shownto be expressed as mRNA in breast tumors as well as in normal brain.However, the published sequence was lacking 3′ sequence. Publishedincomplete sequence for NY-BR-1.1 is represented by GenBank accessionnumber AF269088. A recent BlastN search of the GenBank High ThroughputGenomic Sequence database using Ny-Br-1.1 as a query sequence showed a100% match to the working draft sequence of chromosome 9 (GenBankaccession number AL359312), yielding further 3′ DNA sequence forNy-Br-1.1. The compilation of the Ny-Br-1.1 sequence with the additional3′ sequence from chromosome 9 yielded a 3720 bp ORF sequence (SEQ IDNO:576) which encodes a 1240 amino acid protein sequence (SEQ IDNO:577).

Real time PCR primers were designed to a unique region of NY-BR-1.1 todistinguish its mRNA expression profile from B726P. This experimentrepresents relative values, as it was done without template. Thefirst-strand cDNA used in the quantitative real-time PCR was synthesizedfrom 20 μg of total RNA that was treated with DNase I (AmplificationGrade, Gibco BRL Life Technology, Gaithersburg, Md.), using SuperscriptReverse Transcriptase (RT) (Gibco BRL Life Technology, Gaithersburg,Md.). Real-time PCR was performed with a GeneAmp™ 5700 sequencedetection system (PE Biosystems, Foster City, Calif.). The 5700 systemuses SYBR™ green, a fluorescent dye that only intercalates into doublestranded DNA, and a set of gene-specific forward and reverse primers.The increase in fluorescence was monitored during the wholeamplification process. The optimal concentration of primers wasdetermined using a checkerboard approach and a pool of cDNAs from tumorswas used in this process. The PCR reaction was performed in 25 μlvolumes that included 2.5 μl of SYBR green buffer, 2 μl of cDNA templateand 2.5 μl each of the forward and reverse primers for the gene ofinterest. The cDNAs used for quantitative real time PCR reactions werediluted 1:10 for each gene of interest and 1:100 for the β-actincontrol. Levels of mRNA were expressed relative to ureter whereNY-BR-1.1 expression was not observed when compared to the β-actincontrol.

The real time PCR results show that mRNA expression for NY-BR-1.1 ispresent in breast tumors as well as in normal adrenal gland, brain,retina and testis.

EXAMPLE 14 Characterization of B726P Monoclonal and Purified PolyclonalAntibody Epitopes

Mouse monoclonal antibodies and rabbit polyclonal sera were raisedagainst E. coli derived B726P recombinant protein and tested by ELISA asdescribed in further detail below, for antibody epitope recognitionagainst overlapping 20 mer peptides that correspond to the amino acidsequence of the downstream ORF of B726P (B726P dORF, set forth in SEQ IDNO:176, encoded by SEQ ID NO:175). Numerous peptides were recognized bythe monoclonal and polyclonal antibodies. The corresponding amino acidsequences of these peptide antibody epitopes are summarized in Table 3and are set forth in SEQ ID NO:578-593.

ELISA ANALYSIS: B726P recombinant protein and peptides were coated onto96 well ELISA plate: 50 ul/well at 2 ug/ml for 20 hrs at 4 C. Plateswere then washed 5 times with PBS+0.1% Tween 20 and blocked with PBS+1%BSA for 2 hr. Affinity purified B726P polyclonal antibodies were thenadded to the wells at 1 ug/ml and B726P monoclonal supernatants wereadded neat (220A43 and 220A86 were diluted 1/60 and 1/20 respectively).Plates were incubated at room temperature for 30 minutes and then washedagain as above, followed by the addition of 50 ul/well donkeyanti-mouse-Ig-HRP antibody for 30 minutes at room temperature. Plateswere washed, then developed by the addition of 100 ul/well of TMBsubstrate. The reaction was incubated 15 minutes in the dark at roomtemperature and then stopped by the addition of 100 ul/well of 1N H2SO4.Plates were read at OD450 in an automated plate reader. Peptides withOD450 readings three times background or above were considered to bepositively recognized by the corresponding antibody. TABLE 3 Peptidesrecognized by B726P Antibodies B726P Monoclonal Supernatant B726PPurified Polyclonal 220A2.1 220A19.1 220A94.1 220A151.1 220A43 220A86 (1ug/ml) B726P 289-308 225-244, 73-252 145-164, 232-252 145-164, 1-20,9-28, 17-36, 24-44, peptides 232-252 153-172 153-172 97-116, 105-124,113-132, (amino acids) 121-140, 129-148, 137-156

EXAMPLE 15 Analysis of Autoantibodies to B726P In Breast Cancer Sera andEpitope Mapping of the Antigenic Sites

Specific B726P peptide epitopes were identified that react withautoantibodies in the serum of breast cancer patients. Thirty-threeoverlapping peptides were synthesized spanning the entire B726P protein.These 33 peptides were tested in ELISAs to evaluate which epitopesreacted with breast cancer sera. Reactive epitopes were identifiedthroughout the molecule and a total of 16/74 sera samples from breastcancer patients had reactivity with one or more peptides.

Thirty-one overlapping synthetic peptides spanning the entire B726Pdownstream ORF sequence (amino acid sequence set forth in SEQ ID NO:176)were synthesized and 30 of these were tested in ELISA with sera frombreast cancer patients as well as control sera. The amino acid sequencesof the 31 overlapping peptides of the B726P downstream ORF are set forthin SEQ ID NO:594-624. Three additional peptides of B726P, set forth inSEQ ID NO:625-627 were also tested. Several peptides thoughout themolecule showed reactivity, in particular peptide #2735 (amino acids31-50; SEQ ID NO:597), peptide #2747 (amino acids 151-170; SEQ IDNO:609), peptide #2750 (amino acids 181-200; SEQ ID NO:612), peptide#2753 (amino acids 211-230; SEQ ID NO:615), and peptide #2766 (aminoacids 231-250; SEQ ID NO:617). A total of 16/74 breast cancer sera werereactive with at least one peptide.

B726P antibody epitopes were also mapped using rabbit antisera generatedagainst the B726P downstream ORF (SEQ ID NO:176). The epitopesidentified using the rabbit antisera were as follows: peptide #2732(amino acids 1-20; SEQ ID NO:594), peptide # 2733 (amino acids 11-30;SEQ ID NO:595), peptide #2742 (amino acids 101-120; SEQ ID NO:604),peptide #2743 (amino acids 111-130; SEQ ID NO:605), peptide #2744 (aminoacids 121-140; SEQ ID NO:606), peptide #2745 (amino acids 130-151; SEQID NO:607), peptide #2751 (amino acids 191-210; SEQ ID NO:613), andpeptide #2753 (amino acid 211-230; SEQ ID NO:615). Some low levelreactivity was observed for peptide #2772 (amino acids 291-310; SEQ IDNO:623) and peptide #2773 (amino acids 298-317; SEQ ID NO:624).

The above results confirm that B726P can be used alone or in combinationwtih other breast tumor antigens as a vaccine target. Additionally,these results show that detection of antibodies to B726P can be used asa diagnostic indicator of breast cancer either alone or in combinationwith detection of antibodies to other antigens (e.g. Her-2/Neu or otherantigens known to be expressed in breast cancer tissue).

EXAMPLE 16 Immunohistochemical Analysis of B726P Expression InMetastatic Breast Cancer

Affinity purified polyclonal antibodies anti-B726Pdown (generatedagainst the B726P downstream ORF) were used to assess B726P proteinexpression in metastatic breast cancer samples.

In order to determine which tissues express the breast cancer antigenprotein B726P immunohistochemistry (IHC) analysis was performed on adiverse range of tissue sections. Tissue samples were fixed in formalinsolution for 12-24 hrs and embedded in paraffin before being sliced into8 micron sections. Steam heat induced epitope retrieval (SHIER) in 0.1 Msodium citrate buffer (pH 6.0) was used for optimal staining conditions.Sections were incubated with 10% serum/PBS for 5 minutes. Primaryantibody (rabbit affinity purified anti-B726Pdown) was added to eachsection for 25 minutes followed by 25 minute incubation with anti-rabbitbiotinylated antibody. Endogenous peroxidase activity was blocked bythree 1.5 minute incubations with hydrogen peroxidase. The avidin biotincomplex/horse radish peroxidase (ABC/HRP) system was used along with DABchromogen to visualize antigen expression. Slides were counterstainiedwith hematoxylin to visualize cell nuclei.

Anti-B726Pdown immunoreactivity was observed in 7 of 10 metastaticbreast cancer samples analyzed but not observed in various normaltissues including normal breast. Thus, these data confirm earliermicroarray analysis (see Example 1) showing that B726P is overexpressedin breast tumor tissue as compared to normal tissue. Therefore, thisantigen may be used in diagnostic and immunotherapeutic applications forbreast cancer.

EXAMPLE 17 Analysis of B726P Expression Using Immunoprecipitation andWestern Blot Analysis

Affinity purified polyclonal antibodies generated against the B726Pdownstream ORF protein set forth in SEQ ID NO:176 (anti-B726Pdown) wereused to assess the protein expression of the combined ORF of B726P inbreast cancer cell lines as compared to normal cells as described below.Since the combined ORF includes both the upstream and downstream ORFs,the antibodies generated against the downstream ORF crossreact with thecombined ORF polypeptide as set forth in SEQ ID NO:475.

Cells were lysed in 1% Triton lysis buffer on ice for 10 minutes.Lysates were centrifuge at 15000 rpm and supernatant was saved forimmunoprecipitation (IP)/Western analysis. 2 μg of anti-B726downpolyclonal antibody was added to the supernatant and rocked overnight at4° C. 20 μl of protein G bead slurry was added and incubated for 1 hour.Beads were then washed 3 times with 1 ml of lysis buffer. LDS samplebuffer and β-mercaptoethanol were added and the samples were heated for5 min at 95° C. Samples were size fractionated by gel electrophoresis,transferred to nitrocellulose and Western blotted with the mouseanti-B726down monoclonal antibody A2.1.

³⁵S methionine labeling/IP analysis was carried out as follows: Cellswere grown in 10% Fetal Bovine Serum (FBS) media to desired density.Cells were then starved with DMEM lacking methionine containing 0.1% FBSmediafor 10-15 minutes. FBS was added to a final concentration of 10%along with ³⁵S-Methionine trarislabel (300 μCi −1 mCi). After incubatingfor 3-4 hours the cells were harvested, washed, and lysed. B726P wasimmunoprecipitated as described above and samples were size fractionatedby gel electrophoresis before being exposed to autoradiography film.

The results from the above described experiments showed that the fulllength 148 kDa form (also called NYBR1), the 110 kDa combined ORF form,and the 35 kDa downstream ORF form are all expressed in breast tumorcell lines HTB21 and BT474 but not in the SKBR3 normal breast cell line.Therefore, these results confirm that these forms of the B726P proteinare expressed in breast tumor cell lines and not in normal cells.

U.S. patents, U.S. patent application publications, U.S. patentapplications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification and/or listedin the Application Data Sheet, are incorporated herein by reference intheir entirety.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. An isolated polypeptide comprising an amino acid sequence selectedfrom the group consisting of: (d) sequences set forth in SEQ IDNO:594-627; (e) sequences having at least 70% identity to a sequence setforth in SEQ ID NO:594-627; and (f) sequences having at least 90%identity to a sequence set forth in SEQ ID NO:594-627.
 2. An expressionvector comprising a polynucleotide encoding a polypeptide of claim 1operably linked to an expression control sequence.
 3. A host celltransformed or transfected with an expression vector according to claim2.
 4. An isolated antibody, or antigen-binding fragment thereof, thatspecifically binds to a polypeptide of claim
 1. 5. A method fordetecting the presence of a cancer in a patient, comprising the stepsof: (a) obtaining a biological sample from the patient; (b) contactingthe biological sample with a binding agent that binds to a polypeptideof claim 1; (c) detecting in the sample an amount of polypeptide thatbinds to the binding agent; and (d) comparing the amount of polypeptideto a predetermined cut-off value and therefrom determining the presenceof a cancer in the patient.
 6. A fusion protein comprising at least onepolypeptide according to claim
 1. 7. A composition comprising a firstcomponent selected from the group consisting of physiologicallyacceptable carriers and immunostimulants, and a second componentselected from the group consisting of: (a) polypeptides according toclaim 1; (b) polynucleotides encoding a polypeptide according to claim1; (c) antibodies according to claim 4; (d) fusion proteins according toclaim 6; (e) antigen presenting cells that express a polypeptideaccording to claim
 1. 8. A method for stimulating an immune response ina patient, comprising administering to the patient a composition ofclaim
 7. 9. A method for the treatment of a cancer in a patient,comprising administering to the patient a composition of claim
 7. 10. Adiagnostic kit comprising at least one antibody according to claim 4 anda detection reagent, wherein the detection reagent comprises a reportergroup.